IVF实验室及相关重要区域的空气质量控制

Sandro C. Esteves, MD, PhD Director, ANDROFERT

Andrology & Human Reproduction Clinic Campinas, BRAZIL

IVF实验室及相关重要区域的空气质量控制

TQM Summit, CHINA 2014

目录

ANDROFERT androfert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC REFERRAL CENTER FOR MALE REPRODUCTION PROF. DR. SANDRO ESTEVES 2013 OCTOBER






ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION SC ESTEVES, 2 2014 March

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空气质量对IVF的重要性

监管机构: 要求

我们是如何实施AQC

我们的经验

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空气质量在IVF的重要性动物/实验性研究证据

Study Population Method Outcome

Little and Mirkes, 1990

In vitro cultured rat embryos

Protein and DNA analysis

High levels of acrolein associated with embryotoxicity

Cohen et al, 1997

Analytical measurement procedure

Air sampling and VOC determination in human IVF labs

Higher levels of VOC in HEPA-filtered lab ambient air and incubators compared with outside ambient air

Hall et al, 1998

Analytical measurement procedure and bioassay

Air sampling and VOC determination in human IVF laboratories; acrolein bioassay using 2-cell mouse embryos

Increased levels of VOC in ambient air. Reduction in aldehyde levels by air filtration using carbon-activated and permanganate. In vitro mouse embryo development, implantation and post-implantation outcome inversely correlated with acrolein levels

Worrillow et al, 2002

Analytical measurement procedure

Design of a high velocity air control system and laboratory with VOC air filtration

IVF laboratory and procedure rooms qualified as class 100 areas. No VOCs found above >0.1 parts per billion



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空气质量在IVF的重要性人类观察性研究证据

Study Population Method Outcome

Mayer et al, 1999

RCT of infertile IVF couples

Embryo culture in incubators with and without VOC filtration

Higher PRs when embryos were cultured in incubators with VOC filters

Racowsky et al, 1999

Infertile IVF couples, observational

IVF and embryo culture in labs and incubators with and without VOC filtration

Reduction in miscarriage in cycles performed in labs and incubators with carbon-activated filters

Boone et al, 1999


Construction of cleanrooms for IVF activities

Reduction in air particles associated with an increase in the number of high-quality embryos

Worrillow et al, 2002


IVF in clean rooms with VOC filtration. Outside and inside ambient air monitored for 2 years

Seasonal correlation between temperature/humidity of outside ambient air serving the IVF air system and VOC levels with impact on IR

Knaggs et al, 2007


IVF lab redesigned to meet the EU directive

Implantation and pregnancy rates increased after the move into the cleanroom

空气质量对IVF的重要性-人类实验的观察研究

CPR Miscarriage

rate

36.9%

23.0%

47.1%

15.0%

Severe Male Factor Infertility (N=399)

Standard IVF lab

P=0.01

P=0.03

Esteves et al. Fertil Steril 2004 Esteves et al. Fertil Steril 2006



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空气质量对IVF的重要性

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ü 无空气过滤的IVF实验室存在高浓度VOC

ü 空气过滤系统可有效地过滤颗粒物和VOC

ü 鼠胚发育与VOC呈负相关

ü 安装空气过滤系统的IVF实验室可帮助临床获得更好的结局（妊娠率、种植率和流产率）



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监管机构的要求

ANDROFERT ANDROFERT

androfert.com.br ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION SC ESTEVES, 7 2014 March

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Region (directive)

European Union (EU directive 2004/23/EC; 2006/86/EC)

Brazil (Anvisa RDC33/2006; RDC23/2011)

Particle filtration

Equivalent to GMP Grade A air quality in the critical areas with a background environment at least equivalent to Grade D (exceptions apply)

At least equivalent to ISO class 5 (NBR/ISO 14644–1) in the critical areas

Microbial contamination

Microbial colony counts equivalent to those of Grade A as defined in the current GMP guide with a background environment at least equivalent to Grade D

Microbiological monitoring required; specifications not defined

Volatile organic compounds filtration

Not required

Ventilation systems should be equipped with filters imbedded with activated charcoal



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当科学研究发现了可能的风险，即使还

没有达成科学共识；

我们也有社会责任来保护公众健康免受

暴露于有害物质的风险。

预防原则

*Convention on Biological Diversity, UN 1992; Ratified by 192 countries and the EU, except USA, Andorra and South Sudan



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污染任何可能会影响最终产品质量的不良的材料或物质

1. 尘埃清洁剂残留物、衣物纤维、皮屑（短

时悬浮粒子），1至100µm

2. 微生物病毒，孢子，细菌,（长时悬浮粒子），

1 至 8µm

污染物是由实验室人员，流程，家具和设备所产生的



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污染物

3. 室内挥发性有机化合物VOC 任何可挥发的并与室内空气发生反应的化

合物

产生亚微颗粒和有害副产物（甲苯、苯、醇类、酮类、醛类、制冷气体）

香烟烟雾、汽车尾气、清洁剂、润滑剂、染料、家具、橡胶、塑料、树脂、合成

纤维、尼龙、碎料板、胶合板



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生殖实验室VOC含量

Indoor air

Personnel

Material and Equipment

Cell, tissue and fluids

5-fold increase in VOC

Anderson et al. 1997



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通常病人看到是…

…AND NOT WHAT IS BEHIND THE DOORS!

我们是如何实施AQC

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规划和构造

现场湿式作业低VOC材料表面和涂层照明设备平装和封闭式弓形接合不锈钢和铝材（无VOC）家具，门，窗，排气口，工作站



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空气处理通风系统



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如何避免对配子和胚胎的污染

细菌和其他污染物能自行吸附在尘埃颗粒上。所以减少尘埃颗粒可以改善空气质量

粉尘（粗）过滤器: >10µ 细微粉尘过滤器: 1<10µ

高效空气净化器 : 0,3µ = 99,97%

加压流动空气并通过一系列过滤器以便更高效去除空气中的微粒



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Particle size/m3 of air: ISO 14.644-1 0.3 µM 0.5 µM 1 µM 5 µM

ISO 1 ISO 2 10 4 ISO 3 102 35 8 ISO 4 1,020 352 83

IVF lab ISO 5 10,200 3,520 832 29 ISO 6 102,000 35,200 8,320 293

OR ISO 7 352,000 83,200 2,930 Transfer ISO 8 3,520,000 832,000 29,300

ISO 9 35,200,000 8,320,000 293,000

洁净室分级

VOCs是比HEPA过滤器的有效孔径小100-1000倍的化合物

碳颗粒之间的云离域电子充当电子胶水去除化学污染物

高锰酸钾可通过氧化降解醇类和酮类化合物

如何避免对配子和胚胎的污染

使用正空气加压强制空气流动并通过一系列的含有活性炭和高锰酸钾的过滤器去除VOCs



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如何监测



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监测指标仪器

气体体积流量温差式风速仪气体交换率温差式风速仪

大气-室内气压差微风速仪、辐射热测定器

HEPA过滤器完整性烟雾发生器

尘埃粒子计数电子微粒计数仪

还原能力烟雾发生器

光照水平照度计

噪音水平分贝计

温湿度温度计、湿度计

Third-part certification company (CCL, Brazil); semi-annually IEST 006.2 standards



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VOCs检测

活性碳寿命估计四氯化碳活性法

应用Tenax TA吸附剂和2,4二硝基苯的活性取样苏玛罐

热解析气相色谱法

(Limits < 2 µg/m3)



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沉降培养皿霉菌和血琼脂培养皿

表面采样拭子和接触培养皿

手套印迹

培养箱，工作站，空气，地板，墙壁

菌落形成单位(CFU) 沉降培养皿

（直径90mm) CFU/4 小时

接触培养皿(直径55mm)

CFU/皿

手套印迹CFU/手套

<3 <3 <3

微生物检测



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Esteves & Bento. RBM Online 2013

ü 经核准通过标准



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我们的收获

ANDROFERT ANDROFERT


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空气质控 & IVF 结局 2,315个病人; 14,660 个胚胎

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

Before After 2002-‐2004

After 2005-‐2007

After 2008-‐2010

TQE D3 (%)Miscarriage (%)LBR fresh (%)



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平均每个病人可多获得一个胚胎用于移植或冷冻

空气质控 & IVF 结局 2,315 patients; 14,660 embryos



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IVF累计妊娠率

ANDROFERT ANDROFERT ANDROFERT

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ANDROFERT Esteves, 38 ANDROFERT, Referral Center for Male Reproduction

ET #3 (FET) 49

ET #2 (FET) 239

ET #1 (fresh) 822

50.5% +18.8%

+25.0% Female Age ≤38

AN

DR

OF

ER

T

332/822 63/239 17/49

结论 (1) 动物和人类研究显示空气质量（颗粒物和VOC）与胚胎

体外发育有关

监管机构已出台IVF实验室与空气质量有关的具体指南和要求

我们建立了IVF洁净室技术（胚胎学和男科学实验室，手术室，胚胎移植室）并显示了它的可行性



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结论 (2)

7年间，我们的ART技术在洁净室环境下进展良好

洁净室技术的费用:

仪器: 150,000美元运作: 15,000美元/年

过滤器，洁净室器具/材料/保养，认证

每个病人增加的费用： 130美元/周期



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Health & Medicine

IVF实验室及相关重要区域的空气质量控制