修改MicroSecure玻璃化：一种安全，简便，高效的冷冻程序人胚泡

Summary

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Abstract

临床胚胎玻璃化冷冻与发展，在21 ^世纪 ，并与误解，独特的玻璃化设备，在“开放式”系统（即直接LN ₂接触）的超快速冷却是必不可少的，以优化玻璃化成功的发展。周围的冷却速率的重要性的教条导致受技术变化和创造玻璃化设备的不安全做法忽视重要的质量控制因素（ 例如，易用性，可重复性，可靠性，标签安全和存储安全）。了解允许旨在最大限度地减少区域内和跨技术变化的安全，可靠，可重复和可靠μS-VTF方法的开发其它设备的质量控制瑕疵。同样重要的是，它结合了两种现有符合FDA标准的设备的可用性：1）0.3毫升的离子交联聚合物树脂胚胎吸管内化，双色，篡改-P屋顶标签可重复密封焊缝潜力;和2）缩短，常用，300微米的ID无菌flexipettes直接加载胚胎（多个），以便创造一个高度有效的全球玻璃化设备。像其他无菌，封闭玻璃化系统（ 例如，高安全性玻璃化（HSV），快速-i和VitriSafe）生殖医学有效利用，microSecure玻璃化（μS-VTF）已经证明，它可以实现高变暖后的生存和怀孕结果凭借其注重简单性，并降低技术变化。虽然含有内疏水塞0.3毫升胚胎秸秆市售用标准精液吸管具有棉聚乙烯吡咯烷酮（PVP）插头取代，它保持了其离聚物树脂组合物，以确保焊接密封。但是，棉塞可以在接触时灯芯出flexipettes的流体胚胎内容。一种改进μS-VTF方法适用于包括附加的内部焊接在设备负载侧的插头前密封。增加的技术步骤，将μS-VTF程序并没有影响它的成功应用，为高存活率（> 95％）和妊娠率今天仍在继续。

Introduction

玻璃化是一个最有影响力的辅助生殖技术的体外受精 （IVF）行业自内单精子注射的发展。今天，囊胚是没有事先与传统的慢速冷冻方法¹相关的胚胎活力的丧失冻存。凭借可靠的气候变暖后的胚胎存活率，不孕不育行业转变成首选用冷冻胚胎移植周期，其产生类似的或更高的怀孕结果比传统的新鲜胚胎移植。胚泡活检和胚胎植入前遗传学筛选（PGS）协会，玻璃化已经成为一个重要的临床工具，通过整倍体单胚胎移植^2，3，优化健康的活产结果。

小鼠胚胎玻璃化冷冻是在80年代中期开发的^4， </SUP> ⁵和⁶ 1990年适用于畜牧业。基于的前提是，玻璃化的解决方案，形成一个亚稳glasseous状态，自由损坏冰晶形成的，它已被证明更有效地保护胚胎的完整细胞完整性。有趣的是，有前途的玻璃化验收进入人体胚胎中才开始实现，直到21 ^世纪 。早期出版物推广使用玻璃化恰逢独特的“开放式”系统设备^7，8，9的开发。然而，采用玻璃化到临床实践是缓慢的，因为它是在一个时候缓慢冷冻囊胚进行了改进，还发生。成功的常规慢速速率冷冻，除了玻璃化，用在胚胎培养系统的改进对准，以及与incorporat的囊胚压扁办法离子，这增强了滋养层的两个总体存活，随后注入^10。

在过去十年中，玻璃化技术已迅速取代常规冷冻做法。在很大程度上，这是由于专业玻璃化设备的开发。一些设备已经通过引入内在设计缺陷在IVF行业¹¹所使用的设备阻碍临床玻璃化的整体安全性，效率和效益。事实上，不同的设备之间的细微差别引入程序之间显著技术变型中，通常被称为“技术签字^”12。因此，科学期刊，如可视化杂志实验（朱庇特），可以作为证明的技术细节，这将有助于减少结果差异的宝贵资源。另一项正在进行的问题在于S青梅胚胎学家继续被误导，即使在今天的基础上声称，在“开放的玻璃化系统”胚胎或卵母细胞的“超快速冷却（即用液氮（LN _2）直接胚胎接触）是优化的前提条件成功率“。显然，这种信仰是不准确的基础上，无菌的成功经验封闭系统^{13，14，15。}

基于玻璃化的cryobiological原理，玻璃化的功效比上冷却速率^16，17，18更高度依赖变暖率。在一般情况下，独立于所使用的玻璃化装置的，在暖速率必须超过的冷却速度，以确保高的存活率。高变暖速率尽量减少任何冰的增长（ 即 RECR机会期间变暖的失透相在低温溶液核杂质）ystallization。理所当然的，玻璃化溶液（ 即，类型和所用冷冻保护剂的浓度）的稳定性可能有混淆的效果，但是这是在一个单独的出版物¹¹寻址。考虑到冷却速度变暖问题，MicroSecure玻璃化（μS-VTF）在2008年被开发为一种廉价，非商业，FDA标准的方法优化玻璃化的质量控制方面的问题。这是因为它提供了防篡改，内化，双色标记是唯一的。此外，通过装载和直接在用于移液无菌flexipette存储胚胎（即没有吸移到辅助装置表面），并通过使用使用自动缩放器完全焊封，技术变化已经有效地消除离聚物树脂吸管。

在评估的C为潜在用途的玻璃化设备ompleteness，有应考虑几个质量控制的因素，包括：1） 标注电位 -可以标签牢固附着？他们在防篡改？他们提供了双颜色标识的潜力？是否需要一个次要的标签，并可以在标签保存记录（ 即病人的验证）气候变暖后很容易地删除？ 2） 技术轻松 -能胚胎很容易装入/到设备，及时，简单地识别和跟踪升温后？ 3） 程序简单/重复性 -Does玻璃化方法提供简单，可靠，可以轻松地允许重复性，其中技术人员（内部）和项目（外部）之间的变化降到最低？ 4）LN ₂的存储容量 -可以在设备上轻松和安全地处理，并确定了？是他们的申通快递风靡全球的潜在空间效率？从是否物理损坏或可能的污染物如无菌封闭系统的设备提供安全保障？ 5） 恢复潜力/生存能力 -Is器件设计容易出现胚胎的保证回收潜在的问题，并能可靠地玻璃化并保持完整的细胞的完整性后变暖？后者具体质量的关注，回收率，实际上已令人惊奇地最小化在已发表的报告;这是由通常藏身于一般好的生存率不利的结果（ 即，丢失胚胎或卵子）完成的。任何容易出现不一致的恢复时间（<99％）的设备存在严重缺陷，并构成一个程序的责任。

我们的无菌，封闭μS-VTF方法已发展战略性考虑到每个质量控制措施。然而，5年内优良临床成功和确认¹⁴之后，程序不得不吨Ø修改。原0.3毫升胚胎吸管（具有疏水塞）从体外受精行业中除去，并用0.3毫升精液吸管具有一个标准的棉/ PVP插头（ 即，重新标记为精液/胚稻草）取代。该程序文件概述安全，简单和有效地执行μS-VTF所需要的具体步骤和策略。此外，我们强调需要可靠地占供给的限制的变形（多个），直至作为替代理想吸管容器重新放回临床实验室。

Protocol

The development of the µS-VTF procedure was conducted as part of an approved Institutional Review Board (IRB) study in 2008-2009 (Aspire IRB, Santee, CA) on human oocyte and embryo cryopreservation. The procedure has since been routinely applied to the clinical treatment of infertility patients who have signed informed consent forms. 1. Quality-control Considerations Use different colors (e.g., pens, labels, or identification (ID) rods or plugs) to distinguish patients if cryopreserving more than one group of embryos in a day. Use different dishes and pipettes for each patient. Minimize the volume of medium transferred when moving the blastocysts from one solution to another. Cryopreserve one blastocyst per straw or device. Maintain aseptic/sterile technique and adhere to all safety precautions. Confirm the Physicians' Referral Form and signed patient consent before initiating the procedures. Check the disposition of the patients' embryo(s) in inventory. Use a "Release of Liability and Consent for Transport" if the blastocysts are to be transferred to another facility. Ensure that all signatures are witnessed onsite or notarized. Confirm proper quality-control measures for the medium and the protein supplements, as appropriate, including bioassays, endotoxin determinations, and expiration/lot number verification and tracking. Establish critical limits (i.e., low levels of concern) for clinical cryopreservation outcome monitoring and evaluation, such as recovery rates: < 95%, survival rates: < 80%, and clinical pregnancy rates using single-euploid blastocysts: < 50%. 2. Cryopreservation Procedure Preparation Complete the appropriate paperwork, including entering data into the worksheets and the cryopreservation database and inventory log(s). Prepare fresh cryopreservation solutions or use a commercial source (abiding by the manufacturer's recommended storage and shelf-life conditions). Straw preparation. Open the labeling side of the individualized sterile packets. Detach the filling nozzle found on the traditional 0.3-mL embryo straw by grasping it against the packaging and partially lifting and pulling the straw out; this will expose the label end to ambient conditions while the straw rests aseptically inside the plastic package. (Modification) For 0.3-mL semen/embryo straws, push the plug down 0.5 cm. Apply an internal seal in front of the cotton-PVP plug through use of a basic tabletop impulse sealer. Set the straw onto the Teflon surface of the electrode, just in front of the plug. Depress the handle to activate the heat. Release the handle when the red light appears. To ensure a complete weld seal, heat at a temperature that sufficiently flattens the opposing surfaces, then flip it over 180° and reseal the opposite surface19 as needed. Using a cryomarker, label each straw/device and cryogoblet with the patient name, a unique ID number (e.g., SSN, DOB, or patient ID number), and the date of freezing. Also include a unique number on each straw/device (e.g., 1, 2, 3, etc.). Secure the label (preferably colored) onto a color-weighted ID rod to be inserted and sealed into a 0.3-mL embryo straw. Return the straw to its individual sterile package until use. Note: If only 30-mm color-weighted ID rods are available, then the additional insertion of two ball bearings is needed, adjacent to each side of the ID rod. Identify each cane with a unique inscribed ID number. Identify a LN2 storage tank number and a canister number where each patient's specimen(s) can be stored long-term. Prepare fresh cryo dishes (100 mm) by labeling the bottom surface with the patient name, solution ID, and embryo/holding droplet ID (Figure 1A). Specifically, set up 4 rows of droplets: isotonic hepes-buffered medium (top), V1 (equilibration solution (ES)), V2 (intermediate solution (IS)), V3 (vitrification solution (VS); bottom). Write the embryo ID on the upper-right side with labeled columns. Note: A series of 25- to 50-µL wash droplets (n = 3) is used to ensure that each individual final 10- to 15-µL holding droplet/solution type (i.e., V1, V2, or V3) is pure and undiluted. Using this approach, up to five individual embryos can by vitrified using a single dish. Keep each dish covered when not in use to avoid any room temperature evaporation/dehydration of the droplets. Prepare sterile flexipettes by cutting 3 cm off their base end (referred to as "VTF tips"). Insert them onto individual pipettes (set at 3 µL) and set the pipette-VTF tips upright in a styrofoam tube rack (Figure 1B). Embryo Selection Confirm patient/specimen identification. Using an inverted microscope (200 – 400X magnification), grade and classify the blastocysts according to the Gardner grading system20, using a numeric classification of 1 to 6 for early to hatched blastocysts, respectively, and an A, B, or C grade assigned to the inner cell mass (ICM) and trophectoderm (TE). Note: In preparation for blastocyst biopsy and euploidy analysis, the zona pellucida is pre-hatched by laser ablation on Day 3 to promote the early herniation of the TE and thus requires a modified grading classification2. In brief, Grade 3 full blastocysts exhibit up to 10% TE herniation, Grade 4 expanded blastocysts are 10 – 50% hatched, and Grade 5 hatching blastocysts have > 50% TE extrusion (Figure 2A-C). Note: Our preference is to cryopreserve Grade 3 or higher blastocysts of ≥BB quality on Day 5 or 6. However, vitrified blastocysts of lower quality (C) and post-compaction embryos at earlier stages (including late morula to Grade 1 or 2 blastocysts) can certainly survive warming completely intact and yield viable embryos capable of achieving live births. Collapse the blastocoele of each blastocyst prior to the onset of the cryodilution process by micropuncturing a trophectoderm junction or by performing a laser pulse ablation of a trophectodermal cell21 if using low-molarity vitrification solutions (< 6.5 M or 40% v/v). Note: The need to collapse the blastocysts is not device dependent. In our own early development of µS-VTF (2008), we initially used ethylene glycol (EG)/DMSO solutions successfully with oocytes (1 out of 1 full-term pregnancy), but a sub-optimal (< 80%) survival/development of mouse blastocysts16 was experienced by others21. Since high molar glycerol-based solutions (exceeding 7.9 M) are used in our current VTF system (since 2009), physical blastocoele collapse is unnecessary. However, the selective hatching of hatched blastocysts is advisable in general. Cryodilution and Blastocyst Loading Remove the patient culture dish from the incubator and confirm patient/specimen identification with another person (i.e., witnessed by a secondary source) before removing the embryo(s) to be vitrified. Confirm blastocyst development per step 2.2.2, update the cryo data sheet, and, under stereomicroscopy, pipette blastocyst(s) from the culture dish into an isotonic holding droplet of the cryo dish. Move each blastocyst into individual holding droplets using the vitrification flexipette (i.e., the VTF tip). Move each blastocyst into V1 (ES) solution. Prefill the flexipette with V1 solution (3 µL) and place half the contents onto the embryo. Aspirate the embryo and move to the first of three V1 wash droplets (referred to in step 2.1.6), pipette the embryo 2 – 3 times around perimeter, and clear the pipette contents in the droplet (avoiding bubble formation) or outside on the dish surface. Fill the VTF tip with the next V1 wash drop and repeat the aspiration of the embryo(s). Repeat a third time and move the blastocyst(s) to an individual V1 holding drop. The dilution/washing steps should take 30 to 45 s. Pre-load each VTF tip with the next solution (e.g., V2), insert the pipette in a rack, and use the next pipette (according to the setup in step 2.1.7) (Figure 1B). Note: Since the total exposure time in non-dimethyl sulfoxide (DMSO) containing V1 and V2 solutions is 5 min each, the pipetting of individual blastocysts should be evenly staggered within that interval, considering that the final V3 step will require at least 1 min per blastocyst to perform. For example, if there are 4 blastocysts in a vitrification group, pipette blastocysts #1-2-3-4 at 75-s intervals. Note: The Innovative Cryo Enterprises (ICE) dilution protocol described above is distinctly different than most other commercial vitrification procedures, which generally involve DMSO-containing solutions. Those protocols achieve the same stepwise dilutions through a gradual, but less controlled, merging of wash solution (i.e., isotonic medium), ES, and VS. Repeat steps 2.3.4 and 2.3.5 using V2 (IS) solutions. Repeat steps 2.3.4 and 2.3.5 using V3 (VS) solutions. Upon placing each blastocyst in the V3 holding droplet, clear the residual solution and bubbles from the VTF tip (outside the droplet), and then fill the tip completely with clean V3 around the embryo. Expel approximately one-third of the volume (1 µL) and pipette the blastocyst(s) and remaining V3 completely into the VTF tip. Remove the VTF tip from the pipette, wipe the tip dry of residual V3 on the outer surface by using a sterile gauze pad, and insert the tip end-first into the open end of the pre-labeled straw. A "dry tip" is critical to preventing possible inner straw adherence. Invert the straw (label end down), observe the tip at the inner plug or seal, and safely seal the open end with 1 cm of air space. Preferably, use an automatic sealing device (e.g., Syms I) to eliminate technical variation. Note: The advantage of using straws that are composed of an ionomeric resin plastic (e.g., HSV or µS-VTF) is that using any properly-operated heat sealing device creates a reliable "weld" seal, as noted in step 2.1.3.1. Once sealed, plunge the closed straw directly into LN2 in a stainless steel Dewar flask and place the straw(s) into the open goblet attached to the patient's cane for storage. Note: Because µS-VTF straws use 40-mm weighted ID rods to offset the buoyancy of the air-filled straw, the use of inverted goblets or empty cryovials to cover the specimen(s) is not required unless transportation is involved. Warming and Cryo-solution Elution/Dilution Confirm the Physicians' Referral Form regarding vitrified embryo transfer date, scheduled time, and embryo details (number to thaw/transfer, specific embryo number if PGS tested, etc.). Prepare fresh warming solutions (1.0 M sucrose stock solution) and/or use a commercial source (recommended 1 week-1 month of shelf life once in use, 4 °C storage). Aliquot 17.1 g of sucrose into 50-mL sterile flasks upon initial opening (e.g., weekly supply source) due to the risks of endotoxin accumulation in sucrose granules upon repeated ambient exposure. Mix pre-weighed granular sucrose into solution (1.0 M stock = 3.42 g/10 mL of hepes-buffered human tubal fluid [H-HTF]) and warm the solution up to 37 °C to increase the solubility of the granules. Repeatedly invert the container to help speed up and complete the final mixing. Note: Filtration (0.22-µm filter) performed using positive pressure filtration units is recommended for viscous solutions (> 0.5 M sucrose) or high volumes. Hand pressure pumps are sufficient and inexpensive, whereas an electrical pump is noisy, causes vibrations, and is simply not required. Warm (37 °C) one 10-mL tube each of 1.0 M sucrose solution and H-HTF medium per patient for µS-VTF warming bath use. Prepare fresh thawing dishes (6-well plate) by labeling the cover surface with the patient name and the solution IDs. Note: In this case, we used: (1) T1 wash (200 µL without oil overlay, (2) T1 (100 µL) with 1 mL of oil, (3) T2 (100 µL) with oil, (4) T3 (100 µL) with oil, (5) T4 (100 µL) with oil, and (6) T5/200 µL isotonic HEPES-buffered medium with 10% human serum albumin (HSA) or 20% serum substitute without an oil overlay. Apply a universal sucrose solution step-down protocol-T1 = 1.0 M, T2 = 0.5 M, T3 = 0.25 M, and T4 = 0.125 M-if the ICE or another commercial source is not available, as proposed for slow-frozen embryos22. Furthermore, note that the initial T1 wash and the final T5 equilibrations can be performed in 30-mm Petri dishes and the 4-well dish used for steps 2-5 above, involving an oil overlay. Thaw only one patient's embryo(s) at a time. Check the Physicians' Referral Form and confirm the cycle before warming the indicated number (i.e., 1 or 2 blastocysts); assess survival/probable viability by observing osmotic changes (i.e., shrinkage and rehydration) and intact membranes, and, if questionable, contact the physician and/or patient before warming another blastocyst. Confirm patient/specimen identification with a co-worker (i.e., witnessed by a secondary source). Place the patient cane in a Dewar flask filled with LN2 and isolate identify the straw to be warmed. Bring the 6-well dilution dish to the center of the stereomicroscope stage. Placing a 60-mm Petri dish off to one side (i.e., depending on the left-hand or right-hand preference of the technician), add the warmed sucrose (1.0 M) and H-HTF solutions to create a 0.5 M sucrose warming bath (uncovered). Note: VTF tips are only warmed in 0.5 M sucrose solutions as a QC safeguard, to insure the retention of embryo viability in the rare event that an embryo is expelled upon rapid warming22. Hold the upper straw seal above liquid level to confirm the identity, and then grasp and secure the straw below the internal plug using Mayo scissors (the VTF tip should still be submerged in LN2). Firmly tap the scissors on the Dewar flask (a couple times) to remove the VTF tip from the inner straw sidewall as a QC precaution; if the VTF tip is adherent, the tapping ensures that the tip base drops to the sealed end opposite the labeled end, thus providing an air space near the plug end. Lift the straw with the scissors into ambient air in a horizontal position (next to or below the stereoscope surface) and grasp the non-labeled end (label to the right side). Cut the straw at the inner plug or seal and lift it above the warming bath dish. Pour the VTF tip into the warm sucrose bath at a 60° angle until it is completely extruded and has been rapidly warmed (> 6,000 °C/min); the base of the flexipette will rest above the dish sidewall. Allow the VTF tip to free-fall into the bath. Gently tap the upper straw surface if the tip does not immediately emerge. If it only appears part way, assist in the extrusion by grasping the shaft of the flexipette with the scissors or fine forceps. After 5 – 10 s, grasp the pipette base and insert into a disposable pipetting device; a hole in the bulb acts to release pressure upon insertion. Cover the bulb hole with an index finger and gently squeeze the bulb to release the vitrified blastocyst into the T1 wash (#1) while viewing by stereomicroscopy. Once confirmed, clear the residual V3 (VS) solution and bubbles by positive pressure, evacuating the contents into the center, unused discard well. Start a 5-min timer. Note: All sucrose dilution steps are performed at room temperature (21 ± 2 ºC). Remove VTF tip and place it onto a pipette. Proceed by moving the embryo into T1 #2 under oil for the remaining time (approximately 3 – 4 min). If a second blastocyst is required for transfer, immediately warm a second straw and VTF tip (repeating steps 2.4.9 – 2.4.11) from the patient before initiating step 2.4.12. Move both blastocysts together for a minimum elution in T1 (1.0 M sucrose) of at least 3 min. Pre-fill the flexipette with the next solution, and then move and dilute the blastocyst(s) in T2, T3, and T4 at 3-min intervals. If the zona pellucida has not been previously laser ablated (i.e., hatched), do so in T4. Place the dish onto the stage of an inverted microscope and image the embryo on a computer monitor. Align the zona within a designated red circle and activate 2 or 3 impulses of a diode laser (beginning at the edge of the perivitelline space and moving outward) to create an opening2,19. Equilibrate the blastocysts in T5 (i.e., isotonic medium) for 5 min on a warm surface (37 °C). Assess the initial embryo survival based on the predominant presence of intact cell membranes with even, translucent cytoplasms devoid of pyknotic darkening. Place the embryo(s) into culture for 2 – 6 h before embryo transfer, at which time, the embryo development/survival should be re-evaluated and documented. Note: If more than 1 viable blastocyst exists at the time of transfer and the patient opts to proceed with only 1 blastocyst, the supplemental blastocyst can then be successfully re-vitrified (rVTF) by repeating steps 2.3.3 – 2.3.11. We have several confirmed live births following single rVTF events. Note: rVTF has been experimentally performed up to 5 times without a significant effect on the survival/viability of aneuploidy human blastocysts vitrified in a metastable glycerol-based solution.

Representative Results

1,341 vitrified blastocysts were warmed between 2012 to June 2014, and 1,341 embryos recovered (100%) while 1,316 survived (98.1%). Biopsied blastocysts experienced over 99.5% survival. Upon transferring predominantly single, genetically tested, euploid, vitrified embryos, we were able to achieve some of the highest implantation rates and live birth rates in the USA, independent of age (Figure 3)20, according to recent national statistics reported by the Center for Disease Control and the Society for Assisted Reproductive Technologies (Tables 1 and 2). Evaluating a good prognosis patient population (less than 35 years old) for cryopreserved cycles alone (Table 1), our laboratory outperformed the national average for both implantation rates (i.e., the efficiency of an embryo to establish a pregnancy) and ultimately live birth rates by over 30%. Additionally, the initial validation/verification of our modified storage straws in mid-2014 using 70 research-consented, re-vitrified aneuploid blastocysts resulted in 100% recovery and 100% survival. Figure 1. MicroSecure VTF Setup. The VTF dish (A) is assembled with 3 distinct rows of vitrification solutions, which utilize 3 wash droplets before placement in distinct, numbered holding droplets. Additionally, individual pipettes with shortened VTF tips (i.e., 300-µm ID flexipettes) are secured and organized in a styrofoam tube rack (B), which can be rotated for orderly use. Note that a representative disposable micropipette bulb pipetting assembly is resting on the styrofoam rack. Please click here to view a larger version of this figure. Figure 2. Modified Blastocyst Grading System. Our modified Gardner blastocyst grading system accounts for the induced herniation of TE cells to facilitate blastocyst biopsy. The modification considers the premature hatching of full blastocysts (A: Grade 3 = less than 10% herniation) and expanded blastocysts (B: Grade 4 = up to 50% herniation), while a hatching blastocyst (C) has greater than 50% herniation16. Please click here to view a larger version of this figure. Figure 3. Comparative Pregnancy Outcomes. Over a cumulative 1.5-year interval, pregnancy data were compared to assess the effects of transferring vitrified-warmed euploid blastocysts (n = 172 cycles/144 transfers) compared to non-PGS cycles (n = 160 cycles/153 transfers) involving predominantly fresh transfers18. For our experimental purposes, this data clearly reveals that biopsied blastocysts vitrified by the µS-VTF procedure maintain their viability. Please click here to view a larger version of this figure. Data Source # Cycles Mean # of embryos transferred Implantation Rates (%) Live Birth Rates (%) NB Lab * 144 1.2 74.00% 74.50% National Average 20,423 1.7 39.60% 44.10% * Data were averaged based on the 2014 performance of Orange County Fertility, Southern California Fertility Center and the Southern California Center for Reproductive Medicine clinics using the current Ovation Fertility laboratory (NB Lab) which founded the microSecure vitrification procedure in 2008. Table 1. 2014 CDC Assisted Reproductive Statistics. Frozen embryo transfer pregnancy data of women under 35 years old from three reporting physician clinics using our NB Lab compared to the US 2013 national average of over 450 reporting clinics. SART National Averages Clinic – State  Women  Women  Women  Women   < 35 yrs 35-37 yrs 38-40 yrs 41-42 yrs SCCRM-CA * 63.1% 58.3% 40.6% 32.3% CCRM-CO * 64.7% 61.5% 40.4% 32.2% RMA-NJ * 63.2% 59.7% 34.6% 18.7% National Average 48.6% 38.3% 24.3% 12.3%    SCCRM—Southern California Center for Reproductive Medicine; CCRM—Colorado Center for for Reproductive Medicine; and RMA—Reproductive Medicine Associates *  These leading clinics have all predominantly implemented vitrified embryo transfer cycles, in association with preimplantation genetic testing, into their standard practice of patient care to optimize live birth success per embryo transplanted. Table 2. 2014 Cumulative live birth rates, as reported by the Society for Assisted Reproductive Technologies (SART), for the SCCRM Clinic using our Ovation Fertility Lab in Newport Beach, CA, contrasted with several other respected programs, as well as with the SART national averages.

Discussion

Today, there is a high expectation of attaining complete blastocyst survival (> 95%) and achieving implantation success similar to that of fresh embryos. Some groups have suggested that the live birth rates of vitrified embryo transfer cycles are perhaps even higher than fresh blastocysts when the intact cryopreserved embryo is transplanted into a healthy, non-hormonally-stimulated uterus. Our data clearly indicate that vitrification effectively and reliably maintains the viability of the fresh embryo. Furthermore, we have proven that our aseptic, closed method, called MicroSecure Vitrification (µS-VTF), can achieve an optimal outcome comparable to or greater than the commercial standards (i.e., open device systems) used in the IVF industry.

Variation is associated with technical repeatability and reliability between individuals using a multitude of vitrification devices/methods. In turn, this has resulted in inconsistencies between programs applying vitrification. Therefore, it is not surprising that device familiarity is an important factor regarding laboratory proficiency and successful outcomes. It is this concept of "technical signature"¹¹ that explains why repeatability between programs may be problematic. Not only has the development of more than a dozen commercial devices complicated this phenomenon, it has created quality-control concerns. Fortunately, closed vitrification systems, like µS-VTF, Rapid-I, and VitriSafe, are now proving to be equally effective to open device systems^12,13,14.

MicroSecure VTF is a novel, aseptic vitrification technique developed with technical ease, reliability, and cryo-security in mind. By combining the use of two previously-approved, FDA-compliant devices, it is a non-commercial vitrification system with the distinct advantage of having an established low cost, in contrast to specialized devices. In addition, its unique tamperproof and internalized dual-colored labeling system, as well as numerous other quality-control advantages, have made µS-VTF an attractive global option¹¹. As a non-commercial VTF device, however, its widespread industry application is evolving slowly. Only through the continued publication of its safety, security, and clinical effectiveness will µS-VTF gain growing utilization.

In August 2014, when faced with the inability to acquire the original 0.3-mL embryo straw manufactured with an inner, non-wicking hydrophobic plug, we were able to reliably modify the µS-VTF method. In short, the new 0.3-mL semen/embryo straws with their cotton-PVP plugs were effectively adapted. This was simply achieved by creating an inner seal before the cotton plug, thus preventing contact and fluid wicking with the open-ended VTF tip (i.e., the flexipette containing the embryo or eggs). Furthermore, if 40-mm ID rods are not accessible, the buoyancy issue associated with the lighter 30-mm rods can be counterweighted using two ball bearings.

These additional steps have made the technique slightly less simple, but still highly effective. Today, economics and efficacy are becoming increasingly important concerns, as biopsied blastocysts are typically vitrified individually until their euploidy status is confirmed on a genetics report. Blastocysts with a confirmed non-viable aneuploidy status typically get discarded, with patient consent, within weeks. Thus, a majority (> 50%) of VTF devices are discarded after short-term storage, causing an escalating annual cost when using commercial devices. Overall, µS-VTF is a highly-effective, reliable, and repeatable procedure for the cryopreservation of human blastocysts. The superior quality-control design securely labels and safely stores embryos/oocytes while eliminating recovery failure and optimizing post-warming survival and viability, which justifies the system's use as a universal approach to embryo vitrification.

Materials

Aluminum Cane	IVM	XC055
Ball bearings, 3/32"	VXB.com	KIT15977	stainless steel
CBS semen/embryo straw, 0.3ml	CryoBioSystems	25292	individual sterile
Color, ID rods, 30 mm	CryoBioSystems	019021-26	weighted
Culture tubes, 15ml	Falcon	352099	Conical
Culture tubes, 10ml	Falcon	352057	Snap-cap
Cryosleeves	Nalgene	5016-001
Filter, 250ml	Fisher Sci.	09-740-2A	0.22 μm
Flasks, Tissue Culture 50ml	Falcon	353014
Flexipettes, 300μm ID	Cook Med.	K-FPIP-1300-10BS-5	Sterile, 20/pack
Forcep, Large	Miltex	6-30TC
Forcep,Splinter – fine	Miltex	17-305
Goblet	IVM	PA003
Heat Sealer, SYMS 1	CryoBioSystems	16399	110V or 220V with adapter
Hepes-buffered media	Life Global or	LGGH-100; 100ml,  or	stored at 2-8ºC
	Irvine Scientific	H-HTF; 90126; 100ml	with non-essential AA's
Labels, Cryo	GA International	CL-23T1	Various colors
Liquid Nitrogen Tank, 40L	MVE or Taylor Warton	various	liquid storage
LN2 Dewar flask, 0.5L	Hampton Research	HR4-695	Stainless steel
6-well Custer Dishes	Biogenics	015/020	plasticware by case
Pipette Bulb, Micro Cap	Drummond	Fisher#13681451	Hole on bulb apex
Petri Dishes, 35mm	Falcon	351006
Petri Dishes, 58mm	Nunc	150288
Petri Dishes, 100mm	Falcon	351029
Pipette Tips, ART long	Fisher Sci.	02-707-80	10-100μl
Pipet Aid	Drummond or Falcon	various	rechargeable
Pipetting Device, Stripper	Cooper Surgical	MXL3-STR
Pipettes, Serological 1ml	Falcon	357521
Pipettes, Serological 2ml	Falcon	357507
Pipettes, Serological 5ml	Falcon	357543
Pipettes, Serological 10ml	Falcon	357551
Scissors, Surgical Mayo	Miltex	5-SC-16
Stereomicroscope	Nikon, Olympus, Leica	various
Sterile Gauze pads, 4"x4"	Kendall Healthcare	6939
Synthetic serum	Life Global or	LGPS-20 ; 20ml,  or	stored at 2-8ºC
	Irvine Scientific	SS-99193; 12 x 10ml	purchase low endotoxin lot
Sucrose	Sigma Chemical Co.	#S9378	Aliquot into 50ml flasks,  1year
			17.1g/flask +Medium to 50ml
			makes a 1M solution
			Filter with 0.22µm unit
Timer	Nalgene	5016-001
Thawing  Solution	Innovative	BL-TS	T1, T2, T3, T4
	Cryo Enterprises	(≤1.0M Sucrose)	stored at 2-8ºC for ≤1 month after opening
Vitrification  Solution*,**	Innovative	BL-VS	V1, V2, V3
	Cryo Enterprises	(≥7.9M [Glycerol/EG])	stored at 2-8ºC for ≤1 month after opening
			* Non-permeating cryoprotective additives may include: sucrose, ficoll and sodium hyaluronate
			** other commercial preparations are typically ethylene glycol (EG)/dimethyl sulfoxide (DMSO; 30% v/v; 4.8M),  but could be EG/propylene glycol (32% v/v; 5.2M).  Mixed solutions are typically used to reduce cryo-toxicity concerns of a high molar solution.  Commercial solutions typically include an ES and VS solution. The formulation of commercial preparation is typically proprietary property.