精子畸形率对体外受精-胚胎移植治疗结局的影响

目的回顾性分析我中心接受常规体外受精（IVF）及卵胞浆内单精子注射（ICSI）治疗中,男方精子畸形率对受精率、胚胎质量及临床结局的影响。方法选取本中心2008年9月至2010年5月接受IVF的344对及ICSI的178对夫妇,分为常规IVF组和ICSI组,组内按照男方精子畸形率分为正常形态组（IVF266／ICSI76）和畸精子症组（IVF78／ICSI102）。受精后分别统计IVF及ICSI两组内畸精子症组和正常形态组正常受精率、优质胚胎率、种植率、临床妊娠率及流产率的差别。结果在IVF中,畸精子症组和正常形态组的正常受精率、种植率、临床妊娠率及流产率分别为64．32％／60．09％、33．78％／37．02％、42．03％／54．62％及12．5％／4．23％。两组间受精率无显著性差别,畸精子症组的临床妊娠率显著性低于正常形态组,而早期流产率显著高于正常形态组（P〈0．05）;ICSI组中,畸精子症组和正常形态组的正常受精率、种植率、临床妊娠率及流产率分别为68．01％／64．59％、32．26％／33．78％、43．75％／52．63％及4．76％／5％。畸精子症患者的临床妊娠率较正常组显著性降低（P〈0．05）。将两种受精方式的畸精子症组间比较,IVF的患者早孕流产率显著高于ICSI者（P〈0.05）。结论常规IVF中畸精子症不影响正常受精。对于畸精症子患者,其临床妊娠率均较精子形态正常组低,但是采用ICSI治疗可以显著降低早孕流产率。‘     

A retrospective comparison of pregnancy outcome following conventional oocyte insemination vs intracytoplasmic sperm injection for isolated abnormalities in sperm morphology using strict criteria

Initial in vitro fertilization (IVF)-embryo transfer studies found poor fertilization and pregnancy rates following conventional fertilization of oocytes when using sperm with <4% normal morphology using strict criteria. Some consider today that sperm with only < or =5% normal morphology using strict criteria are associated with infertility. However, other studies have disputed the diagnostic potential of low strict morphology in identifying subnormal male fertility. Based on the original studies most IVF centers perform intracytoplasmic sperm injection (ICSI) when the sperm shows low morphology using strict criteria to allow selection of normal sperm. However, ICSI adds extra time for the embryologist and extra expense for the infertile couple. The present study retrospectively compared fertilization, pregnancy, and implantation rates according to the 2 methods of oocyte fertilization with sperm having normal morphology using strict criteria of < or =5% in women < or =39 years. All fresh embryo transfers were performed on day 3. There was a significantly higher fertilization rate with ICSI. However, there were significantly higher clinical and delivered pregnancy and implantation rates following conventional insemination. The rate of canceled transfers due to no available embryo was 1.9% with conventional insemination vs 1.5% for ICSI in women with adequate egg reserve. Hopefully, this retrospective study will generate interest in a prospective study.     

异常形态精子对植入前胚胎发育和妊娠结局的影响

目的探讨异常形态精子(畸形率≥98%)对植入前胚胎发育及妊娠结局的影响。方法采用回顾性队列研究,分析2017年1～12月在唐都医院妇产科生殖医学中心行ART助孕的2 419例患者临床资料,根据异常形态精子分为3组,即IVF对照组(畸形率≤96%,n=2 129)、IVF实验组(畸形率≥98%,n=90)和ICSI实验组(畸形率≥98%,n=200)。比较3组间植入前受精失败率(受精率<30%)、正常受精率、可用胚胎率等胚胎发育参数和着床率、临床妊娠率、流产率及活产率等妊娠结局的差异。结果 (1)胚胎发育结果:组间比较,IVF实验组受精失败率显著高于IVF对照组(P<0.05),ICSI实验组的受精失败率为0;ICSI实验组正常受精率显著高于IVF对照组和IVF实验组(P<0.05);IVF实验组可用胚胎率显著低于IVF对照组和ICSI实验组(P<0.05)。(2)妊娠结局:单因素分析结果显示,与IVF对照组、ICSI实验组相比,IVF实验组的着床率、临床妊娠率、流产发生率和活产率差异均有统计学意义(P<0.05);IVF对照组和ICSI实验组组间妊娠结局指...     

The clinical and neonatal outcomes after stimulation of immotile spermatozoa using SperMagic medium

To evaluate the efficiency and safety of SperMagic medium on stimulating the immotile spermatozoa in testicular sperm extraction (TESE) and absolute asthenozoospermia, 96 patients with TESE and 106 patients with absolute asthenozoospermia were enrolled in this study. The motile spermatozoa were detected in 47 TESE patients and 68 absolute asthenozoospermia and these patients were assigned to control group. The immotile spermatozoa in 49 TESE patients and 34 absolute asthenozoospermia were stimulated with SperMagic medium. Patients were treated by standard intracytoplasmic sperm injection (ICSI). There were no significant differences in fertilisation, cleavage, implantation, pregnancy, live birth and neonatal outcomes. SperMagic medium does not increase incidence of adverse neonatal outcomes and is a reliable tool for selection of viable spermatozoa in ICSI.     

Intracytoplasmic sperm injection outcomes with cryopreserved testicular sperm aspiration samples

Intracytoplasmic sperm injection (ICSI) may be performed with testicular frozen–thawed spermatozoa in patients with nonobstructive azoospermia (NOA). Sperm retrieval can be performed in advance of oocyte aspiration, as it may avoid the possibility of no recovery of spermatozoa on the day of oocyte pickup. There are few studies available in the literature concerning the use of frozen–thawed spermatozoa obtained from testicular sperm aspiration (TESA). To evaluate the effects and the outcomes of ICSI with frozen–thawed spermatozoa obtained by TESA, we performed a retrospective analysis of 43 ICSI cycles using frozen–thawed TESA. We obtained acceptable results with a fertilisation rate of 67.9%, an implantation rate (IR) of 17.1%, and clinical and ongoing pregnancy rates of 41.9% and 37.2% respectively. The results of this study suggest that performing ICSI using cryopreserved frozen–thawed testicular spermatozoa with TESA as a first option is a viable, safe, economic and effective method for patients with NOA.     

精子DNA完整性与体外受精-胚胎移植临床结局的相关性研究

目的 探讨精子DNA完整性与精液参数及体外受精-胚胎移植（IVF-ET）/卵胞浆内单精子注射（ICSI）临床结局的关系.方法 选择2008年6月～2009年6月在解放军105医院生殖医学中心接受IVF/ICSI治疗的179对不育夫妇作为研究对象,采用吖啶橙试验（AOT）对116例实施IVF和63例实施ICSI治疗的男性患者进行精子DNA完整性分析,根据精子DNA碎片指数（DFI）将患者分为DFI≤30%组和DFI＞30%组,比较两组间精液参数、受精率、卵裂率、优胚率、胚胎冷冻率、着床率和临床妊娠率.结果 DFI ＞30%组精子畸形率显著高于≤30%组（P＜0.01）,但两组间精子密度、活动率、前向运动精子（a＋b）均无显著性差异（P＞0.05）;DFI＞30%组IVF和ICSI的优胚率、ICSI的胚胎着床率和临床妊娠率均显著低于DFI≤30%组（P＜0.01,P＜0.05）.结论 精子DNA完整性与精子形态密切相关,精子DNA损伤在IVF/ICSI过程中对胚胎质量有负面影响,并显著影响ICSI的胚胎着床率和妊娠率,建议行ICSI前应对精子DNA完整性进行评估.     

常规体外受精失败卵母细胞行早期补救卵胞浆内显微注射的临床应用价值

目的探讨常规体外受精-胚胎移植（IVF—ET）中受精障碍患者行早期补救卵胞浆内单精子注射（ICSI）的可行性。方法回顾性分析我院生殖中心2007年1月至2009年7月常规IVF—ET治疗877周期,其中2008年3月至2009年7月期间开展短时受精并对受精障碍周期行早期补救ICSI的546周期作为研究组,2007年1月至2008年2月期间未开展短时受精、早期补救ICSI的331周期作为对照组。研究组通过IVF后6h观察卵母细胞是否排出第2极体评估受精,对完全未受精和低受精周期中未见第二极体排出的成熟卵母细胞立即行早期补救ICSI,比较两组临床及实验室指标。研究组中行早期补救ICSI的70周期实验室指标和临床指标与同期179个常规ICSI周期相比较。结果研究组通过早期补救ICSI,受精率、周期冷冻率、优质胚胎率均比对照组显著提高（P〈O．01）,因受精失败取消移植率显著降低（P〈O．01）。早期补救ICSI周期受精率、可移植胚胎率、临床妊娠率及胚胎种植率与常规ICSI相似,正常受精率、卵裂率和优质胚胎率显著低于常规ICSI,≥3原核（PN）异常受精的比率较常规ICSI略有升高,但无显著性差异。结论IVF后6h行早期补救ICSI能提高常规IVF卵的利用率,并获得与常规ICSI相似的临床妊娠结局。     

ICSI outcome in patients with high DNA fragmentation: Testicular versus ejaculated spermatozoa

Sperm DNA fragmentation (SDF) has emerged as an important biomarker in the assessment of male fertility potential with contradictory results regarding its effect on ICSI. The aim of this study was to evaluate intracytoplasmic sperm injection (ICSI) outcomes in male patients with high SDF using testicular versus ejaculated spermatozoa. This is a prospective study on 36 men with high‐SDF levels who had a previous ICSI cycle from their ejaculates. A subsequent ICSI cycle was performed using spermatozoa retrieved through testicular sperm aspiration. Results of the prior ejaculate ICSI were compared with those of the TESA‐ICSI. The mean (SD) SDF level was 56.36% (15.3%). Overall, there was no difference in the fertilization rate and embryo grading using ejaculate and testicular spermatozoa (46.4% vs. 47.8%, 50.2% vs. 53.4% respectively). However, clinical pregnancy was significantly higher in TESA group compared to ejaculated group (38.89% [14 of 36] vs. 13.8% [five of 36]). Moreover, 17 live births were documented in TESA group, and only three live births were documented in ejaculate group (p < .0001). We concluded that the use of testicular spermatozoa for ICSI significantly increases clinical pregnancy rate as well as live‐birth rate in patients with high SDF.     

Factors from damaged sperm affect its DNA integrity and its ability to promote embryo implantation in mice

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P < .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.     

梗阻性无精子症患者睾丸抽吸精子行ICSI后的胚胎结局

目的总结非手术精子抽吸(NSA)联合ICSI对梗阻性无精子症患者的治疗意义,并探讨精子来源等对ICSI后胚胎结局的影响。方法回顾性分析642个ICSI周期,比较了睾丸抽吸精子和精液精子行ICSI后的受精率、优质胚胎率、临床妊娠率、种植率及胚胎停育率等。结果NSA睾丸精子组和精液精子组相比较,其2PN受精率、优质胚胎率、临床妊娠率、种植率及胚胎停育率之间均没有显著性差异(71．4％vs 73．2％, 73．1％vs 70．1％,34．2％vs 32．6％,22．3％vs 19．8％,21．3％vs 25．0％,P>0．05)。结论非手术抽吸睾丸精子联合ICSI是治疗梗阻性无精子症的一种有效方法。     

Does a microfluidic chip for sperm sorting have a positive add‐on effect on laboratory and clinical outcomes of intracytoplasmic sperm injection cycles? A sibling oocyte study

The most recent technologies for sperm sorting involve microfluidics. However, the most important question whether their use is of any advantage in terms of laboratory and clinical IVF/ICSI outcomes still remains controversy. In this study, we aimed to investigate whether a microfluidic sperm sorting device (Fertile Plus^®) has a positive add‐on effect on laboratory and clinical outcomes. Sibling oocytes of 81 patients were assigned to two sperm sorting groups including swim up and Fertile Plus^®. All embryos were cultured until day 5/6. Fertilisation, embryo quality and blastocyst development were assessed as primary outcomes among 81 patients; clinical pregnancy, implantation and live birth rates were analysed as secondary outcomes as a subgroup analysis due to transfer cancellations. No statistically significant differences were found between groups in terms of all outcomes analysed in laboratory and clinical terms (p > .05 for all). The results of this study suggest that sorting spermatozoa through Fertile chip does not improve laboratory outcomes significantly and does not seem to have a positive contribution to clinical outcomes.     

低剂量阿司匹林在辅助生育技术中的应用

目的　探讨在进行体外受精 -胚胎移植 ( IVF-ET)或卵母细胞浆内单精子注射 ( ICSI)患者口服低剂量阿司匹林对治疗结果的影响。　方法　 IVF或 ICSI治疗的 3 2 4个 ET周期 ,分为是否服用阿司匹林两组 ,分别统计患者注射人绒毛膜促性腺激素 ( h CG)时子宫内膜厚度及 ET胚胎数 ,比较两组的胚胎种植率、临床妊娠率及流产率 ,并进行统计学处理。　结果　两组在注射 h CG日内膜平均厚度、ET中优质胚胎的比率及流产率均无显著性差异 ;未服用阿司匹林组的 ET胚胎数明显多于服用组 ( P<0 .0 1 ) ,而前者的种植率、临床妊娠率却明显低于后者 ( P<0 .0 1 )。　结论　低剂量阿司匹林在 IVF-ET或ICSI的辅助生殖治疗中能有效地提高胚胎种植率 ,增加患者的临床妊娠率     

中国乙型肝炎和非肝炎男性间的精液质量和辅助生殖技术治疗结果的比较

本研究旨在确定乙型肝炎病毒（HBV）感染对精子质量和辅助生殖技术治疗结局的影响。分析2008年1月至2009年12月于浙江大学附属妇产科医院寻求辅助生育治疗的916例男性（457例乙型肝炎病毒携带者和459例阴性对照男性）的精液参数;将2004年1月至2009年12月期间的男性乙肝病毒携带的587个常规体外受精（In vitro fertilisation,IVF）周期和325个单精子卵细胞浆内注射（Intracytoplasmic sperm injection,ICSI）周期作为阳性观察组,根据取卵日期、女方年龄及ART受精方式通过统计软件以1：1比例匹配,设立夫妇双方均为HBsAg阴性的对照组（587个IVF周期,324个ICSI周期）,分析两组间的辅助生殖治疗结局。与对照组相比,乙肝携带者表现有较低的精液体积、精子总数以及较差的精子活动力和形态学表现（P〈0．05）;乙肝男性夫妇接受ICSI治疗后,双原核（2PN）受精率、优质胚胎率、胚胎着床率以及临床妊娠率均显著性降低俨〈0．05）;而两组病人间的IVF治疗结局差异无显著性（P〉0．05）。通过Logistic回归分析发现乙肝病毒感染作为一个独立的因素增加少弱精子症的发生以及降低ICSI治疗周期的胚胎着床率和临床妊娠率（P〈0．05）。我们的研究结果提示了男性乙型肝炎病毒感染与较差的精子质量和ICSI治疗结局有关,而对IVF治疗结局没有明显的影响。     

Observational retrospective study of Intracytoplasmic sperm injection outcomes of 117 infertile men with severe short tail sperm defect

Intracytoplasmic sperm injection (ICSI) is increasingly used to treat male-factor infertility when sperm parameters are not proper for intrauterine insemination (IUI) or in vitro fertilization (IVF). Among sperm abnormalities, short tail sperm defect is a rare kind of teratozoospermia, which is a severe cause of male infertility. In this study, we evaluated the ICSI outcomes of infertile men with severely short tail sperm defect. 117 infertile men with primary infertility were included in this study. We evaluated the impact of short tail sperm defect on large ICSI series (228 cycles) outcomes. The fertilisation rate (FR) was 49.0%, the clinical pregnancy rate (PR) was 21.7%, and the delivery rate (DR) was 17.5%. The results of statistical analysis show that there is no relationship between short tail sperm defect and clinical pregnancy. According to the present study, there were patients with successful ICSI outcomes despite the severe defect in their spermatozoa flagella. Our results can be considered in two main aspects: (a) it seems that ICSI could be a proper therapy for infertile men with short-tailed sperm defect and (b) the abnormal sperm morphology (especially in sperm flagellum) is not a reliable predictor for the ICSI outcomes. In conclusion, our study suggests that ICSI should be considered as a proper treatment way for infertile men with severe short tail sperm defect and probably other sperm flagella abnormalities.     

ICSI中不同来源精子对临床结局的影响

目的:分析不同来源精子在卵细胞胞质内单精子注射(ICSI)后的临床结局。方法:将682个ICSI治疗周期分为:射出精子组(598例)、经皮附睾精子抽吸术(PESA)得到精子组(58例)、睾丸精子获取术(TSE)得到精子组(26例),比较三组的受精率、临床妊娠率、种植率、流产率、异位妊娠率以及分娩率之间的差别。结果:TSE组受精率明显低于射出精子组和PESA组(81.06%vs87.95%,87.82%,P<0.05);射出精子组、PESA组和TSE组的妊娠率(39.46%,48.28%,34.62%)、种植率(19.80%,23.80%,18.34%)、流产率(13.13%,17.86%,11.11%)、异位妊娠率(5.51%,7.14%,11.11%)、分娩率(32.11%,36.21%,26.92%),均没有显著差异(P>0.05)。结论:虽然TSE来源精子对ICSI受精率有所影响,但射出精子和手术(TSE和PESA)来源精子对妊娠结局没有影响。     

Focus on intracytoplasmic morphologically selected sperm injection （IMSI）. a mini-review

Intracytoplasmic sperm injection （ICSI） is the recommended treatment in many cases of male-factor infertility. Several studies have demonstrated a positive correlation between optimal sperm morphology and positive ICSI outcomes. In fact, spermatozoa with severe abnormalities of the head are well documented to be associated with low fertilisation, implantation and pregnancy rates. However, a spermatozoon which is classified as ＇normal＇ by microscopic observation at low magnification could contain ultrastructural defects that impair both the fertilisation process and embryonic development. The intracytoplasmic morphologically selected sperm injection （IMSI） procedure changed the perception of how a spermatozoon suitable for injection should appear. Sperm selection is carried out at x 6000 magnification, allowing improved assessment of the sperm nucleus. Currently, standardized clinical indications for I MSI are lacking and the candidates are selected on the grounds of their medical history or of a careful analysis of the sperm suspension. Further prospective randomized studies are needed to confirm the advantages of IMSI in specific groups of patients. In addition to providing a brief overview of the IMSI procedure, this study aims to review the literature, which explains the theoretical basis and the clinical outcomes of this technique. Several reports show that IMSI is associated with improved implantation and clinical pregnancy rates as well as lower abortion rates when compared to ICSI. Although a possible correlation between the sperm＇s abnormal nucleus shape, increased DNA fragmentation and negative laboratory and clinical outcomes has been long investigated, the results are conflicting.     

Comparison of conventional in vitro fertilisation and intracytoplasmic sperm injection outcomes in patients with moderate oligoasthenozoospermia

The method of choice for assisted reproductive technology treatment in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) is usually based on the evaluation of male infertility factors. Decisions for couples with moderate oligoasthenozoospermia (OA) are often empirical because uniform treatment criteria are lacking. This study aimed to evaluate the effect of patients with moderate OA treated with conventional IVF and ICSI. A total of 199 couples with moderate OA undergoing their first IVF/ICSI cycle were included in the study. The patients were divided into two groups according to the type of insemination: conventional IVF group (n = 97) and ICSI group (n = 102). All patients were randomised to be inseminated either by conventional IVF or ICSI. The fertilisation rate, embryo quality, implantation rate and clinical pregnancy rate were examined. No differences in the fertilisation, implantation and pregnancy rates were observed between conventional IVF and ICSI groups (P > 0.05). However, the number of good‐quality embryos was significantly higher in the ICSI group than in the IVF group (P < 0.05). Couples with moderate OA did not influence on the overall clinical outcomes between IVF and ICSI treatments, and a negative influence by ICSI on blastocyst development was not confirmed.     

Intracytoplasmic sperm injection in the treatment of male infertility due to obstructive or non-obstructive azoospermia

<正> Objective:To evaluate the effects of intracytoplasmic sperm injection (ICSI) ontreatment of infertility due to obstructive and non-obstructive azoospermia.Methods:A retrospective analysis of fertilization,cleavage,embryo implantationand pregnancy rates was done in 158 ICSI cycles including 112 obstructive azoospermiaand 46 non-obstructive azoospermia.Ovarian hyperstimulation and ICSI procedureswere performed by conventional protocol.The sperm was collected by percutaneous epi-didymal sperm aspiration (PESA) or testicular sperm extraction (TESE).Results:The fertilization rate (73.1% vs.67.0%),cleavage rate (88.6% vs.86.3%),embryo implantation rate (20.7% vs.11.4%),clinical pregnancy rate per trans-fer cycle (35.7% vs.19.6%) were obtained for obstructive and non-obstructiveazoospermia,respectively.Conclusion:The results revealed that in the cases of obstructive azoospermia,ferti-lization rate,embryo implantation rate and clinical pregnancy rate were significantlyhigher than those of non-obstructive azoospermia.But there was no significant differ-ence of the cleavage rate between two groups.     

Comparison of intracytoplasmic sperm injection outcomes between spermatozoa retrieved from testicular biopsy and from ejaculation in cryptozoospermic men

The infrequent presence of spermatozoa in cryptozoospermic men ejaculate is a limiting factor in the treatment of them. Sometimes, this consideration impels us to apply meticulous microscopic search in ejaculate or testicular sperm extraction (TESE) method. The aim of this study was to assess putative effectiveness of sperm origin, ejaculated or testicular, in cryptozoospermia treatment. In this context, were evaluated intracytoplasmic sperm injection (ICSI) outcomes in two parameters including fertilisation rate (2PN) and embryo quality, independently. We compared the outcome in two groups: patients who underwent ejaculate/ICSI and ones who underwent TESE/ICSI process. Nineteen ICSI cycles performed with testicular spermatozoa and the rest of cycles (n = 208) carried out with ejaculated spermatozoa. Result analysis showed similar fertilisation rate between testicular and ejaculated spermatozoa (respectively, 60% versus 68%, P ≥ 0.05). Also, on the other hand, embryo quality did not show significant differences between two groups, except grade A with low significance. With regard to almost equal performance of both methods in results and being invasive of TESE as surgical sperm retrieval method, the use of ejaculated sperm more than testicular sperm should be recommended in patients with cryptozoospermia whenever possible.     

Effect of follicle-stimulating hormone on sperm quality and pregnancy rate

Aim: To evaluate the possible links between ultrastructural sperm quality and the clinical pregnancy rate in infertile males treated with FSH before intracytoplasmic sperm injection (ICSI). Methods: Forty-four infertile males with idiopathic oligo-asthenozoospermia were randomly allocated to the treated (n=24) and non-treated (control,n=20) groups. Semen analysis was carried out by light and transmission electron microscopy (TEM) before and 12 weeks after FSH therapy. ICSI was performed in all couples. Results: TEM revealed a significant improvement in sperm quality after FSH treatment, particularly in men with their partners achieving clinical pregnancy. The pregnancy rate was 33% in the treated group and 20% in the control. Conclusion: Results highlight a positive role of FSH therapy in infertile males before ICSI, which was correlated with an increased pregnancy rate in treated couples.We believe that improved sperm ultrastructure after FSH therapy could positively influence the quality and early stage of embryo development, thereby increasing the probability of embryo implantation.