Does recurrent implantation failure exist? Prevalence and outcomes of five consecutive euploid blastocyst transfers in 123987 patients 是否存在复发性植入失败？123987 例患者中连续五次优倍囊胚移植的发生率和结果
Pavan Gill (iD , Baris Ata (iD) , Ana Arnanz , Danilo Cimadomo (iD , Alberto Vaiarelli (iD , Human M. Fatemi (iD , Pavan Gill (iD , Baris Ata (iD) , Ana Arnanz , Danilo Cimadomo (iD , Alberto Vaiarelli (iD , Human M. Fatemi (iD 、Filippo Maria Ubaldi , Juan A. Garcia-Velasco , and Emre Seli (ID Filippo Maria Ubaldi , Juan A. Garcia-Velasco , and Emre Seli (ID ) IVIRMA Global Research Alliance, IVIRMA New Jersey, Basking Ridge, NJ, USA IVIRMA全球研究联盟，IVIRMA新泽西州，美国新泽西州巴斯克里奇市 ART Fertility Clinics, Dubai, United Arab Emirates ART 生育诊所，迪拜，阿拉伯联合酋长国 Department of Obstetrics and Gynecology, Koç University School of Medicine, Istanbul, Turkey 土耳其伊斯坦布尔科奇大学医学院妇产科系 IVIRMA Global Research Alliance, IVIRMA Madrid, Madrid, Spain IVIRMA全球研究联盟，IVIRMA马德里，西班牙马德里 Department of Biomedicine and Biotechnology, University of Alcalá, Madrid, Spain 西班牙马德里阿尔卡拉大学生物医学和生物技术系 IVIRMA Global Research Alliance, Genera-Clinica Valle Giulia, Rome, Italy 意大利罗马瓦莱朱利亚 Genera-Clinica IVIRMA 全球研究联盟 ART Fertility Clinics, Abu Dhabi, United Arab Emirates ART 生育诊所，阿布扎比，阿拉伯联合酋长国 Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA 美国康涅狄格州纽黑文耶鲁大学医学院妇产科和生殖科学系*Correspondence address. IVIRMA Global Research Alliance, IVIRMA New Jersey, 140 Allen Road, Basking Ridge, NJ 07920, USA. E-mail: emre.seli@ivirma.com *通讯地址：IVIRMA 全球研究联盟，IVIRMA 新泽西，140 Allen Road, Basking Ridge, NJ 07920 USA。IVIRMA Global Research Alliance, IVIRMA New Jersey, 140 Allen Road, Basking Ridge, NJ 07920, USA.电子邮件：emre.seli@ivirma.com(D) https://orcid.org/0000-0001-7464-8203 These authors contributed equally to this work 这些作者对这项工作做出了同样的贡献
Abstract 摘要
STUDY QUESTION: What are the clinical pregnancy and live birth rates in women who underwent up to two more euploid blastocyst transfers after three failures in the absence of another known factor that affects implantation? SUMMARY ANSWER: The fourth and fifth euploid blastocyst transfers resulted in similar live birth rates of and , respectively, culminating in a cumulative live birth rate of ( ) after five euploid blastocyst transfers. WHAT IS KNOWN ALREADY: The first three euploid blastocysts have similar implantation and live birth rates and provide a cumulative live birth rate of . STUDY DESIGN, SIZE, DURATION: An international multi-center retrospective study was conducted at 25 individual clinics. The study period spanned between January 2012 and December 2022. A total of 123987 patients with a total of 64572 euploid blastocyst transfers were screened for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a history of any embryo transfer at another clinic, history of any unscreened embryo transfer at participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, untreated intracavitary uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies, adenomyosis, communicating hydrosalpinx, endometrial thickness prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), preimplantation genetic testing cycles for monogenic disorders, or structural chromosome rearrangements were excluded. Ovarian stimulation protocols and embryology laboratory procedures including trophectoderm biopsy followed the usual practice of each center. The ploidy status of blastocysts was determined with comprehensive chromosome screening. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles, natural or modified natural cycles. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 ( of the total population) patients met the criteria and underwent at least one additional euploid blastocyst transfer after failing to achieve a positive pregnancy test with three consecutive euploid blastocyst transfers. Outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers. Overall, the live birth rate was similar with the fourth and fifth euploid blastocysts ( vs , relative risk value ). Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI , cycles using non-ejaculate or donor sperm, double-embryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity assay test result yielded similar results. Where data were available, the fourth euploid blastocyst had similar live birth rate with the first one (relative risk ). The cumulative live birth rate after five euploid blastocyst transfers was ( ). LIMITATIONS, REASONS FOR CAUTION: Retrospective design has its own inherent limitations. Patients continuing with a further euploid embryo transfer and patients dropping out from treatment after three failed euploid transfers can be systematically different, perhaps with regard to ovarian reserve or economic status. 研究问题：在没有其他已知影响着床因素的情况下，妇女在三次失败后再进行最多两次卵裂囊胚移植，其临床妊娠率和活产率如何？简要回答：第四和第五次极性囊胚移植的活产率分别为 和 ，五次极性囊胚移植后的累计活产率为 （ ）。已知：前三个极性囊胚的植入率和活产率相似，累计活产率为 。研究设计、规模和持续时间：在 25 家诊所开展了一项国际多中心回顾性研究。研究时间跨度为 2012 年 1 月至 2022 年 12 月。共筛选出 123987 名患者，共移植了 64572 个单倍体囊胚。参与者/材料、地点、方法：曾在其他诊所进行过任何胚胎移植的患者、曾在参与研究的诊所进行过任何未经筛查的胚胎移植的患者、父母核型异常的患者、使用供体卵母细胞或妊娠载体的患者、未经治疗的腔内子宫病变（如息肉、子宫肌瘤等）患者、使用供体卵母细胞或妊娠载体的患者、使用供体卵母细胞或妊娠载体的患者、未经治疗的腔内子宫病变（如息肉、子宫肌瘤等）患者、使用供体卵母细胞或妊娠载体的患者。不包括：先天性子宫畸形、子宫腺肌症、交流性子宫内膜异位症、开始使用黄体酮前子宫内膜厚度 、男方因非梗阻性无精子症而使用睾丸精子、移植的胚胎染色体拷贝数处于中间水平（即马赛克）、植入前单源遗传检测周期或染色体结构重排。 卵巢刺激方案和胚胎实验室程序（包括滋养层活检）均遵循各中心的惯例。囊胚的染色体倍性状态通过染色体综合筛查确定。子宫内膜准备方案遵循参与中心的惯例，包括计划周期、自然周期或改良自然周期。主要结果和偶然性的作用：共有 105 名患者（占总人数的 ）符合标准，在连续三次优倍囊胚移植均未能获得阳性妊娠试验结果后，他们至少又进行了一次优倍囊胚移植。各参与中心的第四和第五次极性囊胚移植结果相似。总体而言，第四和第五次极性囊胚移植的活产率相似（ vs ，相对风险 值 ）。敏感性分析排除了受精后第 7 天活检的囊胚、体重指数 的女性、使用非射精精子或供精精子的周期、双胚胎移植周期，以及因子宫内膜接受能力检测结果而修改胚胎移植日的周期，结果相似。在有数据的情况下，第四个优倍囊胚与第一个囊胚的活产率相似（相对风险 ）。五次优倍囊胚移植后的累积活产率为 （ ）。局限性、注意事项：回顾性设计有其固有的局限性。 继续进行优生胚胎移植的患者与三次优生胚胎移植失败后放弃治疗的患者可能存在系统性差异，这可能与卵巢储备或经济状况有关。
WIDER IMPLICATION OF THE FINDINGS: Implantation failure seems to be mainly due to embryonic factors. Given the stable and high live birth rates up to five euploid blastocysts, unexplained recurrent implantation failure should have a prevalence of . Proceeding with another embryo transfer can be the best next step once a known etiology for implantation failure is ruled out. 研究结果的广泛意义：植入失败似乎主要是由于胚胎因素造成的。鉴于5个优倍囊胚以内的活产率稳定且较高，原因不明的反复植入失败的发生率应为 。一旦排除了植入失败的已知病因，下一步最好的办法就是再次进行胚胎移植。
STUDY FUNDING/COMPETING INTEREST(s): None. 研究资助/赞助利益：无。
TRIAL REGISTRATION NUMBER: N/A. 试验登记号：无。
Keywords: in vitro fertilization / assisted reproduction / implantation failure / preimplantation genetic testing / live birth 关键词：体外受精/辅助生殖/植入失败/植入前基因检测/活产
Introduction 导言
The term 'recurrent implantation failure' (RIF) aims to define a milestone during the course of multiple embryo transfers, all of which have failed to result in implantation. The recurrent nature of the failures presumably suggests a currently unknown etiology that may affect the success of subsequent transfers, perhaps requiring a different diagnostic and therapeutic approach to achieve a live birth (Ata et al., 2021). Until recently, the term has been used loosely with varying definitions based on female age, developmental stage and quality of embryos transferred, fresh or frozen embryo transfers, as well as number of embryos that failed to implant (Shaulov et al., 2020). The heterogeneity of definitions without an underpinning biological rationale has led to several challenges affecting patients and reproductive medicine professionals worldwide. First and foremost, using low thresholds to diagnose RIF has triggered multiple diagnostic and therapeutic interventions to be offered to couples in the absence of sound biological rationale or proper proof of effectiveness (reviewed in Franasiak et al., 2021). Such an approach does not only consume funds available for further treatment, but also puts couples at risk of unintended harm in the form of complications or lower success rates caused by the new intervention. Second, inclusion of women with too few failed transfers in RIF research leads to heterogenous study populations yielding unreliable and conflicting results and conclusions (Ata et al., 2021). 反复植入失败"（RIF）一词旨在定义多次胚胎移植过程中的一个里程碑，即所有胚胎移植均未成功植入。胚胎移植失败的复发性推测表明，目前未知的病因可能会影响后续移植的成功率，也许需要采用不同的诊断和治疗方法才能实现活产（Ata 等人，2021 年）。直到最近，这一术语的使用还很宽泛，根据女性年龄、发育阶段、移植胚胎的质量、新鲜或冷冻胚胎移植以及未能着床的胚胎数量等因素有不同的定义（Shaulov 等人，2020 年）。在没有生物学依据的情况下，定义的多样性导致了一些影响全球患者和生殖医学专业人员的挑战。首先，使用低阈值诊断 RIF 引发了向夫妇提供多种诊断和治疗干预，但却缺乏合理的生物学依据或适当的有效性证明（Franasiak 等人，2021 年综述）。这种做法不仅消耗了可用于进一步治疗的资金，还使夫妇们面临因新干预措施引起并发症或成功率降低而遭受意外伤害的风险。其次，在 RIF 研究中纳入转运失败次数过少的妇女，会导致研究人群的异质性，从而产生不可靠且相互矛盾的结果和结论（Ata 等人，2021 年）。
There have been recent efforts to define RIF with an underpinning biological rationale (Ata et al., 2021; Somigliana et al., 2022; ESHRE Working Group on Recurrent Implantation Failure, 2023; (The writing group) for the participants to the 2022 Lugano RIF Workshop, 2023). These new approaches recognize embryo aneuploidy as the most common reason for implantation failure, in the absence of other known factors, e.g. communicating hydrosalpinges or intracavitary lesions. Since the incidence of aneuploidy is strongly dependent on female age, recent definitions consider euploidy status of the embryos transferred, either as directly determined by preimplantation genetic testing for aneuploidy (PGT-A) or by using female age to calculate anticipated probability of euploidy. This information is used to estimate the expected cumulative probability of implantation or live birth to be achieved with the number of embryos transferred. If this figure is above a threshold, that is regarded high enough to rule out implantation failure due to randomly occurring aneuploidy, the patient can be diagnosed with RIF (Ata et al., 2021; ESHRE Working Group on Recurrent Implantation Failure, 2023; (The Writing Group) for the Participants to the 2022 Lugano RIF Workshop, 2023). 最近，人们一直在努力根据生物学原理来定义 RIF（Ata 等人，2021 年；Somigliana 等人，2022 年；ESHRE 复发性着床失败工作组，2023 年；2022 年卢加诺 RIF 研讨会与会者（编写小组），2023 年）。这些新方法认为，在没有其他已知因素（如交流性鞘膜积液或腔内病变）的情况下，胚胎非整倍体是植入失败的最常见原因。由于非整倍体的发生率与女性年龄密切相关，最近的定义考虑了移植胚胎的非整倍体状态，即通过植入前非整倍体基因检测（PGT-A）直接确定的非整倍体状态，或利用女性年龄计算预期的非整倍体概率。这些信息可用于估算移植胚胎数达到的植入或活产的预期累积概率。如果该数字高于一个阈值，即足以排除随机发生的非整倍体导致的植入失败，则患者可被诊断为 RIF（Ata 等人，2021 年；ESHRE 复发性植入失败工作组，2023 年；2022 年卢加诺 RIF 研讨会与会者（编写小组），2023 年）。
An initial landmark study reported the cumulative sustained implantation and live birth rates achieved with three consecutive single euploid blastocyst transfers (SETs) in the absence of other obvious factors (Pirtea et al., 2021). Euploid blastocysts had similar sustained implantation and live birth rates across the first three consecutive single embryo transfers, leading to a cumulative probability of implantation and a cumulative probability of live birth among the 4429 women included in the study. These figures were in agreement with the predictions of a mathematical model, which was informed by euploid blastocyst implantation and live birth rates reported in the literature (Ata et al., 2021). Based on these studies, the incidence of 'true' unexplained RIF would be in the IVF population, since a statistically significant progressive decline in either the implantation or live birth rate per euploid blastocyst transferred was not observed between the first three consecutive SETs. 一项具有里程碑意义的初步研究报告了在没有其他明显因素的情况下，连续三次单个极性囊胚移植（SET）所获得的累积持续植入率和活产率（Pirtea 等人，2021 年）。在前三次连续的单胚胎移植中，极性囊胚具有相似的持续植入率和活产率，因此，在参与研究的 4429 名妇女中，植入的累积概率为 ，活产的累积概率为 。这些数字与一个数学模型的预测结果一致，该数学模型参考了文献中报道的优倍囊胚植入率和活产率（Ata 等人，2021 年）。根据这些研究，试管婴儿人群中 "真正的 "不明原因 RIF 的发生率为 ，因为在前三次连续的 SET 之间，没有观察到每移植一个极性囊胚的植入率或活产率出现统计学意义上的显著下降。
Despite significant progress in our understanding of RIF based on these studies, the fate of the fourth and the fifth euploid blastocyst transfers following the failure of the first three remains unknown. If the fourth and the fifth euploid transfers result in implantation with a similar rate as the first three attempts, the true prevalence of RIF could be much . However, if there is a significant drop in the implantation potential of the embryo at the fourth and the fifth transfer, this finding could be used to identify a subgroup of patients with true RIF and possible biological mechanisms preventing implantation. In the current study, we addressed this question by examining clinical pregnancy and live birth rates in women who underwent at least one additional euploid blastocyst transfer after three failures in the absence of another known factor that affects implantation. 尽管基于这些研究，我们对 RIF 的理解有了很大的进步，但在前三次失败后，第四次和第五次极性囊胚移植的命运仍然未知。如果第四和第五次卵裂囊胚移植的着床率与前三次相似，那么 RIF 的真实发生率可能远低于 。但是，如果胚胎在第四次和第五次移植时的植入潜能显著下降，那么这一发现可用于确定真正的 RIF 患者亚群以及阻止植入的可能生物学机制。在当前的研究中，我们针对这一问题，在没有其他已知的影响植入因素的情况下，对三次失败后至少再进行一次优倍囊胚移植的妇女进行了临床妊娠率和活产率的研究。
Materials and methods Study population 材料和方法 研究对象
This was an international multi-center retrospective study conducted at 26 individual clinics operating as part of Reproductive Medicine Associates (RMA) USA, IVI Spain, Genera, Italy, and ART Fertility Clinics, United Arab Emirates (Supplementary Table S1). Research ethics committee approvals were obtained in each participating institution (RMA Network in the USA: Advarra # 00064197; IVI Spain: PI 229/22; Genera Italy 0747/2023-7283; YALE IRB: 2000032741; and ART Fertility Clinics: REFA095-2304ABU-005-BA; DOH/ZHCD/2023/1226). 这是一项国际多中心回顾性研究，在美国生殖医学协会 (RMA)、西班牙 IVI、意大利 Genera 和阿联酋 ART 生育诊所旗下的 26 家诊所进行（补充表 S1）。各参与机构均获得了研究伦理委员会的批准（美国 RMA 网络：Advarra # 00064197；西班牙 IVI：美国 RMA 网络：Advarra # 00064197；西班牙 IVI：PI 229/22；意大利 Genera 0747/2023-7283；耶鲁大学 IRB：2000032741；ART 生育诊所：REFA095-2304ABU-005-BA; DOH/ZHCD/2023/1226).
The study period spanned between January 2012 and December 2022. All patients who failed to achieve a positive pregnancy test after the cumulative transfer of three consecutive euploid blastocysts (regardless of the number of transfer procedures) and who subsequently underwent at least one more euploid blastocyst transfers at participating centers were included. Patients were between the age of 18 and 45 years old and had a BMI . Exclusion criteria included history of any embryo transfer in another clinic, history of any unscreened embryo transfer in participating clinics, parental karyotype abnormalities, the use of donor oocytes or a gestational carrier, intracavity uterine pathology (e.g. polyp, leiomyoma), congenital uterine anomalies (e.g. septum), intramural fibroids distorting uterine cavity, adenomyosis, communicating hydrosalpinx, endometrial thickness prior to initiating of progesterone, use of testicular sperm due to non-obstructive azoospermia in the male partner, transfer of an embryo with a reported intermediate chromosome copy number (i.e. mosaic), PGT cycles for monogenic disorders, or structural chromosome 研究时间跨度为 2012 年 1 月至 2022 年 12 月。研究对象包括所有在参与中心连续移植三次（无论移植次数多少）优生囊胚后妊娠试验未呈阳性，且随后至少再进行一次优生囊胚移植的患者。患者年龄在 18 至 45 岁之间，体重指数 。排除标准包括曾在其他诊所进行过胚胎移植、曾在参与诊所进行过未经筛查的胚胎移植、父母核型异常、使用过供体卵母细胞或妊娠载体、腔内子宫病变（如息肉、子宫肌瘤）、先天性子宫异常（如子宫纵隔、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症、子宫内膜异位症等。子宫腔内病变（如息肉、子宫肌瘤）、先天性子宫异常（如子宫纵隔）、子宫腔内肌瘤扭曲、子宫腺肌病、沟通性输卵管积水、开始使用黄体酮前子宫内膜厚度 、因男方患有非梗阻性无精子症而使用睾丸精子、移植据报告染色体拷贝数为中间值（即马赛克）的胚胎、单基因疾病的 PGT 周期或染色体结构性异常。
rearrangements. Patients who received a double-euploid blastocyst transfer (DET) were excluded if only one of the embryos implanted. 重排。如果只有一个胚胎着床，则排除接受双倍性囊胚移植（DET）的患者。
Participants were stimulated with commonly used ovarian stimulation protocols. Embryology laboratory procedures followed the usual practice of each participating center. Fertilization was achieved with IVF or ICSI depending on sperm characteristics and each center's practice. Embryo culture and trophoectoderm biopsy followed each centers' own protocol. 参与者采用常用的卵巢刺激方案进行刺激。胚胎实验室程序遵循各参与中心的惯例。根据精子的特性和各中心的惯例，采用体外受精或卵胞浆内单精子显微注射进行受精。胚胎培养和滋养层活检按照各中心自己的方案进行。
The ploidy status of blastocysts was determined with comprehensive chromosome screening (CCS), using next-generation sequencing (NGS) (ART, RMA, IVI, Genera) or quantitative real-time polymerase chain reaction (qPCR) (RMA, Genera) (Forman et al., 2013; Scott et al., 2013; Capalbo et al., 2015, 2021; de Los Santos et al., 2018; Tiegs et al., 2021; Ata et al., 2023). CCS diagnoses included euploid, whole chromosome aneuploid, segmental aneuploid, or intermediate copy number (i.e. mosaic). Any transfers of blastocysts with a reported intermediate copy number, or segmental or whole chromosome aneuploid were excluded from the analyses. 囊胚的倍性状态是通过全面染色体筛查（CCS）确定的，采用的是新一代测序（NGS）（ART、RMA、IVI、Genera）或实时定量聚合酶链反应（qPCR）（RMA、Genera）（Forman 等，2013 年；Scott 等，2013 年；Capalbo 等，2015 年、2021 年；de Los Santos 等，2018 年；Tiegs 等，2021 年；Ata 等，2023 年）。CCS 诊断包括超整倍体、全染色体非整倍体、节段非整倍体或中间拷贝数（即马赛克）。任何报告为中间拷贝数、节段性或全染色体非整倍体的囊胚移植都不在分析之列。
Embryos were vitrified after biopsy until time of transfer. Endometrial preparation protocols followed the usual practice of participating centers and included programmed cycles with sequential exogenous estrogen and progesterone administration, natural or modified natural cycles (Vaiarelli et al., 2020; Rafael et al., 2022; Ata et al., 2023). 胚胎在活检后进行玻璃化处理，直至移植。子宫内膜制备方案遵循参与中心的惯例，包括连续使用外源性雌激素和孕激素的计划周期、自然周期或改良自然周期（Vaiarelli 等，2020 年；Rafael 等，2022 年；Ata 等，2023 年）。
Outcome measures 成果措施
The primary outcomes were clinical pregnancy rate and live birth rate after the fourth and the fifth consecutive euploid blastocyst transfer. Clinical pregnancy was defined as the presence of an intrauterine gestational sac and yolk sac. Live birth was defined as delivery of a fetus beyond 22 weeks gestational age (ZegersHochschild et al., 2017). 临床妊娠率和活产率是连续第四次和第五次优倍囊胚移植后的主要结果。临床妊娠的定义是宫内出现孕囊和卵黄囊。活产是指胎龄超过 22 周的胎儿娩出（ZegersHochschild 等人，2017 年）。
Statistical analysis 统计分析
Continuous variables were defined with mean and standard deviation or median and quartiles and compared with KruskalWallis and ANOVA tests depending on distribution characteristics. Categorical variables were defined with numbers and percentages and compared with chi-squared test and its derivatives as appropriate. Unit of analysis was euploid blastocyst transferred. Clinical pregnancy and live birth rates were calculated for the fourth and fifth euploid blastocysts per participating center and in aggregate. Relative risks (RR) and 95% CIs were calculated for comparisons. Sensitivity analyses were done excluding blastocysts biopsied on Day 7 postfertilization, women with a BMI , cycles using non-ejaculate or donor sperm, doubleembryo transfer cycles, and cycles in which the day of embryo transfer was modified due to endometrial receptivity array (ERA) test result. A life table was prepared using the previously published data pertaining to first three consecutive euploid blastocyst transfers and the fourth and fifth euploid blastocyst transfers done in the same center (RMA, New Jersey) with the same CCS platform (Pirtea et al., 2021). 连续变量用均值和标准差或中位数和四分位数定义，并根据分布特征用 KruskalWallis 检验和方差分析检验进行比较。分类变量用数字和百分比定义，并根据情况使用卡方检验及其导数进行比较。分析单位为移植的单倍体囊胚。临床妊娠率和活产率是根据每个参与中心的第四个和第五个优倍囊胚计算得出的。计算比较的相对风险 (RR) 和 95% CI。敏感性分析排除了受精后第 7 天活检的囊胚、体重指数 的女性、使用非射精精子或供精精子的周期、双胚胎移植周期以及因子宫内膜受体阵列（ERA）检测结果而修改胚胎移植日的周期。利用之前公布的前三次连续极性囊胚移植以及在同一中心（RMA，新泽西州）使用相同的 CCS 平台进行的第四和第五次极性囊胚移植的相关数据编制了生命表（Pirtea 等人，2021 年）。
Results 成果
During the study period 123987 patients underwent an egg retrieval and 94401 had an embryo transfer, with a total of 64572 euploid blastocyst transfers performed in the 25 participating clinics. Of the 123987 patients, 105 ( of the total population) underwent at least one more euploid blastocyst transfer after failing to achieve a pregnancy with three euploid blastocyst transfers and met the other inclusion criteria to be included in the analyses. 在研究期间，有 123987 名患者进行了取卵，94401 名患者进行了胚胎移植，25 家参与研究的诊所共进行了 64572 次优倍囊胚移植。在这 123987 例患者中，有 105 例（占总人数的 ）在进行了 3 次优倍囊胚移植后未能成功妊娠，但至少又进行了 1 次优倍囊胚移植，并符合其他纳入分析的标准。
Among the 105 patients, female age was years, BMI was , median serum anti-Müllerian hormone (AMH) level was at the time of first egg retrieval. Male age was years. The origin of the sperm was ejaculate in 99 cycles, fine-needle aspiration due to obstructive azoospermia in one, testicular sperm extraction due to prior vasectomy in one, and donor sperm in four cycles. Thirty-one women had one, 55 women had two, 11 women had three, and 4 women had four oocyte retrieval procedures. 105名患者中，女性年龄为 岁，体重指数为 ，首次取卵时血清抗缪勒氏管激素（AMH）水平中位数为 。男性年龄为 岁。99个周期的精子来源于射精，1个周期的精子来源于梗阻性无精子症，1个周期的精子来源于输精管结扎术前的睾丸取精，4个周期的精子来源于捐精。31名妇女进行了一次取卵，55名妇女进行了两次取卵，11名妇女进行了三次取卵，4名妇女进行了四次取卵。
The median number of oocytes collected was 17 (12-24.5), metaphase two (MII) oocytes was 13 (10-18), fertilization rate per MII oocyte was (73.86-90.91%), blastulation rate per MII oocyte was (33.33-57.14%), and euploidy rate per biopsied blastocyst was (50-83.33%). There were euploid blastocysts per stimulation cycle. Table 1 shows baseline demographics and stimulation cycle outcomes per participating center. 采集到的卵母细胞中位数为 17 个（12-24.5），二分裂期（MII）卵母细胞为 13 个（10-18），每个 MII 卵母细胞的受精率为 (73.86-90.91%)，每个 MII 卵母细胞的囊胚形成率为 (33.33-57.14%)，每个活检囊胚的非整倍体率为 (50-83.33%)。每个刺激周期有 个非整倍体囊胚。表 1 显示了每个参与中心的基线人口统计学和刺激周期结果。
Participants received a total of 493 euploid blastocysts in 461 embryo transfer cycles. All 105 women had failed to achieve implantation after receiving three euploid blastocysts, in two or three embryo transfer procedures. There were 105 first (102 SETs, 3 DETs), 105 second ( 98 SETs, 7 DETs), 105 third ( 99 SETs, 6 DETs), 100 fourth ( 90 SETs, 10 DETs), 33 fifth ( 31 SETs, 2 DETs), 9 sixth ( 9 SETs), 4 seventh (4 SETs), 2 eighth (2 SETs), and 2 ninth (2 SETs) embryo transfer cycles. One-hundred and ninety-four blastocysts were biopsied on the fifth, 265 on the sixth, and 34 on the seventh day postfertilization; 373 blastocysts were tested with NGS and 120 with qPCR. 在 461 个胚胎移植周期中，参与者共接受了 493 个优倍囊胚。所有 105 名妇女都在两次或三次胚胎移植手术中接受了三个优倍囊胚，但都未能成功着床。共有 105 个第一次胚胎移植周期（102 个 SET，3 个 DET）、105 个第二次胚胎移植周期（98 个 SET，7 个 DET）、105 个第三次胚胎移植周期（99 个 SET，6 个 DET）、100 个第四次胚胎移植周期（90 个 SET，10 个 DET）、33 个第五次胚胎移植周期（31 个 SET，2 个 DET）、9 个第六次胚胎移植周期（9 个 SET）、4 个第七次胚胎移植周期（4 个 SET）、2 个第八次胚胎移植周期（2 个 SET）和 2 个第九次胚胎移植周期（2 个 SET）。受精后第五天对 194 个囊胚进行了活检，第六天对 265 个囊胚进行了活检，第七天对 34 个囊胚进行了活检；对 373 个囊胚进行了 NGS 检测，对 120 个囊胚进行了 qPCR 检测。
Endometrial thickness was on the day of progesterone commencement (in programmed cycles), hCG injection (in modified natural cycles), or the day of ovulation (in natural cycles). ERA was used prior to 57 embryo transfer cycles, and in 34, ET day was changed by 1 day based on ERA result. 子宫内膜厚度是在开始使用黄体酮（在程序周期中）、注射 hCG（在修改后的自然周期中）或排卵日（在自然周期中）的当天测量的。在 57 个胚胎移植周期前使用了ERA，其中 34 个周期的胚胎移植日根据ERA结果改变了一天。
The outcomes of the fourth and fifth euploid blastocyst transfers were similar across participating centers and are shown in Table 2. Overall, the live birth rate achieved with the fourth euploid blastocyst was and with the fifth (after four prior failures) was . hCG positivity, clinical pregnancy, and live birth rates were also similar with the fourth and fifth euploid blastocysts as shown in Table 3. 表 2 显示了各参与中心第四和第五次极性囊胚移植的结果。如表 3 所示，第四个和第五个极性囊胚的 hCG 阳性率、临床妊娠率和活产率也相似。
Sensitivity analyses excluding blastocysts biopsied on Day 7 postfertilization (Supplementary Tables S2, S3, and S4), women with a BMI (Supplementary Tables S5, S6, and S7), cycles using non-ejaculate or donor sperm (Supplementary Tables S8, S9, and S10), double-embryo transfer cycles (Supplementary Tables S11, S12, and S13), and cycles in which the day of embryo transfer was modified due to ERA test result (Supplementary Tables S14, S15, and S16) yielded similar results. 敏感性分析排除了受精后第 7 天活检的囊胚（补充表 S2、S3 和 S4）、BMI 的女性（补充表 S5、S6 和 S7）、使用非射精或供精的周期（补充表 S8、S9 和 S10）、双胚胎移植周期（补充表 S11、S12 和 S13）以及因 ERA 测试而更改胚胎移植日的周期（补充表 S11、S12 和 S13）、S9和S10）、双胚胎移植周期（补充表S11、S12和S13）以及因ERA检测结果而更改胚胎移植日的周期（补充表S14、S15和S16），结果相似。
The endometrial preparation protocol was changed for 11 patients ( ), however, without a systematic trend, i.e., from a natural to programmed cycle in one, from a modified natural to a programmed cycle in six, from a programmed to a modified natural cycle in four. Thirty-four ( ) patients underwent the fourth and the fifth euploid blastocyst transfer with same protocol. The distribution of endometrial preparation protocols was similar for fourth and fifth transfers (Supplementary Table S17). The number of patients who underwent six, seven, eight, or nine euploid embryo transfers without achieving a pregnancy was too low for hypothesis testing (Supplementary Table S18). 11例患者（ ）的子宫内膜准备方案发生了改变，但没有系统性趋势，即1例从自然周期改为程序周期，6例从改良自然周期改为程序周期，4例从程序周期改为改良自然周期。34例（ ）患者以相同的方案进行了第四和第五次优胚移植。第四次和第五次移植的子宫内膜准备方案分布相似（补充表 S17）。进行六次、七次、八次或九次极胚胎移植而未妊娠的患者人数太少，无法进行假设检验（补充表 S18）。
There were 24 fourth and 7 fifth euploid single blastocyst transfers from RMA New Jersey, which underwent CCS with NGS 来自新泽西州 RMA 的 24 个第四代和 7 个第五代优倍单囊胚移植接受了 NGS 的 CCS 处理
Table 1. Demographics and stimulation cycle characteristics per participating center. 表 1.各参与中心的人口统计学特征和刺激周期特征。
IVF center groups 试管婴儿中心小组
ART 生殖诊所
ART fertility clinics
意大利属
Genera Italy
IVIRMA 西班牙
IVIRMA Spain
美国 IVIRMA 公司
IVIRMA USA
value 值
Female age in years 女性年龄（岁
0.03
Male age in years 男性年龄（岁
36.8 (13.8)
41.2 (5.6)
40.4 (10.9)
0.02
Serum AMH level in 中的血清AMH水平
0.98
Body mass index in 的体重指数
0.04
Cumulus oocyte complexes 积层卵母细胞复合体
14 (12-20)
15 (10.8-24.3)
18 (12-26.3)
0.78
Metaphase two oocytes 二期卵母细胞
12 (10-15)
16 (10.5-19.3)
0.59
Fertilization rate per metaphase two oocyte 每个二分裂期卵母细胞的受精率
0.14
Blastulation rate per metaphase two oocyte 每个二分裂期卵母细胞的分裂率
0.70
Euploidy rate per biopsied blastocyst 每个活检囊胚的非整倍体率