Embryotoxicity testing of IVF disposables: how do manufacturers test?

Embryotoxicity testing of IVF disposables: how do manufacturers test? Abstract STUDY QUESTION How do manufacturers perform embryotoxicity testing in their quality control programs when validating IVF consumables? SUMMARY ANSWER The Mouse Embryo Assay (MEA) and Human Sperm Survival Assay (HSSA) used for IVF disposables differed from one manufacturer to another. WHAT IS KNOWN ALREADY Many components used in IVF laboratories, such as culture media and disposable consumables, may negatively impact human embryonic development. STUDY DESIGN, SIZE, DURATION Through a questionnaire-based survey, the main manufacturers of IVF disposable devices were contacted during the period November to December 2018 to compare the methodology of the MEA and HSSA. We focused on catheters for embryo transfer, catheters for insemination, straws, serological pipettes, culture dishes and puncture needles used in the ART procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS We approached the manufacturers of IVF disposables and asked for details about methodology of the MEA and HSSA performed for toxicity testing of their IVF disposable devices. All specific parameters like mouse strains, number of embryos used, culture conditions (media, temperature, atmosphere), extraction protocol, subcontracting, and thresholds were registered and compared between companies. MAIN RESULTS AND THE ROLE OF CHANCE Twenty-one companies were approached, of which only 11 answered the questionnaire. Significant differences existed in the methodologies and thresholds of the MEA and HSSA used for toxicity testing of IVF disposables. Importantly, some of these parameters could influence the sensitivity of the tests. LIMITATIONS, REASONS FOR CAUTION Although we approached the main IVF manufacturers, the response rate was relatively low. WIDER IMPLICATIONS OF THE FINDINGS Our study confirms the high degree of heterogeneity of the embryotoxicity tests performed by manufacturers when validating their IVF disposable devices. Currently, no regulations exist on this issue. Professionals should call for and request standardization and a future higher degree of transparency as regards embryotoxicity testing from supplying companies; moreover, companies should be urged to provide the users clear and precise information about the results of their tests and how testing was performed. Future recommendations are urgently awaited to improve the sensitivity and reproducibility of embryotoxicity assays over time. STUDY FUNDING/COMPETING INTEREST(S) This study did not receive any funding. L.D. declares a competing interest with Patrick Choay SAS. TRIAL REGISTRATION NUMBER N/A embryotoxicity testing, human sperm survival assay, mouse embryo assay, IVF disposable, sensitivity Introduction The success of IVF depends on the quality of laboratory procedures, environmental conditions, equipment and reagents, which should be monitored and improved through quality control testing (Gardner et al., 2005, Lane et al., 2008, Morbeck, 2012). Toxicity of supplies used for embryo culture has been well documented and recent reports provide evidence that the quality of supplies is variable (Lierman et al., 2007, Morbeck, 2012, Nijs et al., 2009). Recommendations advocate testing media and consumables before their use due to the potential release of toxic substances which might affect gametes and embryos (Lane et al., 2008, Morbeck, 2012). Indeed, the ubiquitous use of IVF disposable plastics makes them one of the providers of embryotoxic substances (Cohen et al., 1997, DeHaan, 1971, Holyoak et al., 1996, Nijs et al., 2009). Plastics are not usually toxic; however, changes may occur during sterilization, packaging and storage, resulting in the release of breakdown products, such as short hydrocarbon chains, volatile organic compounds (VOC), and in modifications of the electrical charge on their surfaces (Lee, 1999, Sladowski et al., 2008, Vardhan and Shukla, 2018). The toxicity of plastics may also derive from additives (anti-static or anti-viscous agents) which facilitate the transformation of resins and increase their stability (Olivieri et al., 2012). Importantly, embryotoxicity could lead to immediate apoptosis of the gametes and embryos, even after brief contact with the plastic disposable (Nijs et al., 2009, Ray et al., 1987). Thus, embryotoxicity should be suspected in case of decreased fertilization rates or abnormal embryonic development (Nijs et al., 2009), and long-term health consequences in the offspring cannot be ruled out (Morbeck, 2012). Differences in birthweight (Banrezes et al., 2011), imprinting (Fernandez-Gonzalez et al., 2004, Lonergan et al., 2003, Rivera et al., 2008) and gene expression pattern (Ecker et al., 2004) in domestic animal species have been observed when embryos were cultured under sub-optimal conditions. In humans, evidence is accumulating that in vitro culture conditions may influence the perinatal outcomes (Marianowski et al., 2016, Sunde et al., 2016, Zandstra et al., 2018). Moreover, embryotoxicity is dose-cumulative, in the sense that a component with minimal intrinsic toxicity could see its toxicity amplified when combined with other stress factors (Awonuga et al., 2013, Wale and Gardner, 2016). Faced with the in vitro chemical threats, the embryo uses different defence mechanisms, requiring large amounts of energy which hampers development. In this aspect, it is, therefore, essential to check the possible toxicity of IVF consumables before implemented into IVF procedures. Currently, two main bioassays are used to assess the safety of reagents and materials used for IVF. The main test is the mouse embryo assay (MEA) (Ackerman et al., 1984, Ackerman et al., 1985, Fleetham et al., 1993, Parinaud et al., 1987) which involves culturing one-cell or two-cell mouse embryos (Ackerman et al., 1985) up to the blastocyst stage in a culture medium, previously in contact with the consumable being tested. The subsequent blastulation rate is used as a parameter of possible toxicity. A second bioassay is the human sperm survival assay (HSSA) (Bavister and Andrews, 1988, Claassens et al., 2000, Critchlow et al., 1989). This test measures the survival (or the motility) of human sperm after the exposure to the consumable, comparing it to those of non-exposed sperms at specific observation times. The result is expressed as the sperm motility index (SMI) which is calculated by the ratio of the sperm motility of the sample tested divided by the motility of the control. Currently no toxicity testing regulations exist for manufacturers of IVF disposables—only guidelines. In the USA, the current good manufacturing practice of the US Food and Drug Administration (FDA) requires that toxicity tests like MEA should be carried out in culture media and consumables used for IVF without the methodology being specified (FDA, 1998). Similarly, in the EU, manufacturers must demonstrate safety and effectiveness of their products to obtain a CE mark (Medical Device Directive MDD, 1993, MEDDEV 2.7/1 revision 4); however, the mode is not detailed. Thus, there are no well-defined safety and quality standards for ART devices, focussing on safety of gametes, embryos, and offspring (Wetzels et al., 2010). The aim of the present study was to explore the methodology employed for MEA and HSSA testing in quality control programs by the main manufacturers of IVF consumables. Materials and Methods Study design Between November and December 2018, a questionnaire was forwarded to the main manufacturers and distributors of consumables used for IVF in order to determine the methodology used for MEA and HSSA. The questionnaire was also sent to three subcontractors. The products studied were catheters for embryo transfer, catheters for insemination, straws, serological pipettes, culture dishes and puncture needles used in the IVF procedures. Each company was asked to answer the questionnaire (one answer per type of device). In case of no-reply, companies were approached either by telephone or by repeated mail in which case up to five reminder messages were forwarded. Questionnaire Thirty-four questions, 21 on the MEA and 13 on the HSSA, were included in the questionnaire. The following data were recorded for the MEA: mouse strain, number of embryos used for testing, type of embryo (one- or two-cell), origin of the embryos (fresh or frozen), culture conditions (culture medium, micro-drops or wells, individual or grouped culture, %O2, temperature, air quality). Manufacturers were asked about possible subcontracting, the type of products used for testing (raw materials or final products), the number of items tested per lot and the certification of their quality control programs. Moreover, questions included the extraction protocols, the objectives and the accepted toxicity thresholds. Finally, manufacturers were asked if they performed a cell count of the trophectoderm and intra-cellular cell mass (ICM) of blastocysts obtained from the MEA (methodology, number of studied blastocysts, objectives and thresholds). For the HSSA, manufacturers were questioned about subcontracting, air quality during testing, categories and number of tested devices per lot, objectives and the accepted toxicity thresholds. Moreover, the following data were recorded: the extraction protocol, the type of counting technique (manual or automated), the origin of the sperm sample (fresh or frozen), the sperm sample concentration, the type of sperm selection (gradient, swim-up, washing), the medium used, the incubation temperature, the number of sperms counted and the type of motility evaluated (total and progressive). Results In total, 21 companies were approached. Eleven companies answered the questionnaire. Two companies declined to answer due to ‘confidentiality problems’. Three companies replied that they were only distributors and that the manufacturer should be approached directly. Five companies never replied despite five attempts to reach them via either phone or mail. One of three subcontractors answered the questionnaire, one declined to participate and the last subcontractor did not reply despite five reminder messages. MEA The findings of the MEA are summarised in Table I. Among the 11 companies who answered the questionnaire, nine subcontracted their MEA. However, the methodology and the objectives used were not necessarily similar. One company would subcontract, depending on the geographic location of its factories. Another did not subcontract at all. When subcontracted, the tests met the International Organization for Standardization (ISO) 17025 standard and Good Laboratory Practice for Nonclinical Laboratory Studies, Title 21 Code of Federal regulations (CFR) Part 58 Accreditation Program for testing. MEA questions and answers. Table I MEA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) MEA: Mouse Embryo Assay ISO: International Organization for Standardization HTF®: HTF® (Human Tubal Fluid) Medium (Biocare, Rome, Italy) Global®: Global® (LifeGlobal Group LCC, Guilford, USA) BSA: bovine serum albumin EmbryoMax® Modified M16®: EmbryoMax® Modified M16 Medium (Sigma-Aldrich, Saint Louis, USA) EmbryoMax® KSOM®: EmbryoMax® KSOM (potassium-supplemented simplex optimised medium) Medium (Sigma-Aldrich, Saint Louis, USA) Open in new tab Table I MEA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) MEA: Mouse Embryo Assay ISO: International Organization for Standardization HTF®: HTF® (Human Tubal Fluid) Medium (Biocare, Rome, Italy) Global®: Global® (LifeGlobal Group LCC, Guilford, USA) BSA: bovine serum albumin EmbryoMax® Modified M16®: EmbryoMax® Modified M16 Medium (Sigma-Aldrich, Saint Louis, USA) EmbryoMax® KSOM®: EmbryoMax® KSOM (potassium-supplemented simplex optimised medium) Medium (Sigma-Aldrich, Saint Louis, USA) Open in new tab MEA testing Two companies performed MEA on both raw materials and final products, nine companies on the finished products, only. MEA was performed on packed and sterilized products without pre-stage ‘off gassing’ except for one company which opened packages and let them air overnight prior to MEA testing. The number of items tested per lot varied according to the company and the type of consumable, from 1 to 10 per lot, with one company performing MEA on 10 items every 3 lots. Six manufacturers performed MEA tests using hybrids from inbred strains B6C3F1xB6D2F1. One company used strain B6/CBA and another used strain FVB/NxCD1. For two other companies, several different strains of mice were used according to the item tested and the specificity of the requests. A minimum of 15 embryos were used, sometimes as many as 30 or 31. All companies performed MEA with a control group, but the number of embryos used could differ between the test group and control group. Randomization of the embryos was not performed by all. Eight companies used one-cell embryos, and three companies used two-cell embryos. Most embryos were fresh or occasionally a pool of fresh and frozen embryos. Culture media and culture Eight companies used a culture medium for human IVF and two companies used a culture medium for murine IVF. The majority of MEA tests were performed in batched culture. Two, 3, 5, 7 or 21 embryos were dispensed per micro-drop or well, the volume of medium varied from 12.5 μL to 1 mL. Culture was usually carried out at 37 ± 1°C in 5 ± 1% CO2. No test was carried out under hypoxic conditions. The air quality of the laboratory in which the tests were carried out was monitored by one company, only. Two companies answered that the air quality was not monitored; the other eight companies chose to not answer this question. The extraction protocol used for MEA varied according to the manufacturer and the type of devices tested. Examples of extraction protocols on embryo transfer catheters and IVF dishes are presented in Table II. Examples of extraction protocols used by the different companies for MEA or HSSA. Table II Examples of extraction protocols used by the different companies for MEA or HSSA. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. MEA: Mouse Embryo Assay HSSA: Human Sperm Survival Assay Open in new tab Table II Examples of extraction protocols used by the different companies for MEA or HSSA. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. MEA: Mouse Embryo Assay HSSA: Human Sperm Survival Assay Open in new tab MEA objectives The MEA objectives were different among companies (Table I). Three companies used a cut-off of 75% or 80% blastulation rate within 96 h. Six companies added morphological criteria and used a cut-off of 70% or 80% expanded blastocysts rate within 72 or 96 h of culture. Two companies used a cut-off of 80% cleavage rate within 24 h associated to a cut-off of an 80% expanded blastocyst rate within 96 h, or a cut-off of an 80% hatching blastocyst rate within 120 h of culture. Moreover, some companies recorded differences in morphology between the test and control groups. Three companies carried out cell counting of the ICM and trophectoderm by fluorescent microscopy for all blastocysts obtained from the MEA, and the number of cells obtained per blastocyst should not be significantly different from that of the control group. HSSA The findings of the HSSA are summarised in Table III. In contrast to the MEA, the HSSA was not carried out systematically in all consumables used for IVF, but only on items that came into direct contact with sperms, such as insemination catheters, serological pipettes, and some of culture dishes. One manufacturer did not perform the HSSA due to lack of standardization and certification of the test. Six companies subcontracted the test. However, the methodologies and objectives used were not necessarily similar. Usually, a single item was tested per lot, with re-testing performed on a second article if the first test was negative. HSSA questions and answers. Table III HSSA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) HSSA: Human Sperm Survival Assay SMI: sperm motility index Open in new tab Table III HSSA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) HSSA: Human Sperm Survival Assay SMI: sperm motility index Open in new tab Sperm and incubation Mostly fresh donor sperm was used; however, the initial and final concentrations of sperm used were rarely described. Likewise, the sperm preparation protocol was rarely described, usually being either gradient or swim-up. The incubation temperature used was 32 or 37°C, and sperm preparations were sometimes incubated under 5% CO2 in different types of media. As for MEA, the extraction protocol used for HSSA was different among companies. An example of insemination catheters is provided in Table II. Some manufacturers evaluated the total motility, others the progressive motility or both, mostly after 24 h of incubation. One company measured motility after 48 and 72 h incubation. Another company recorded motility at 0, 4, 8 and 24 h but used the 8-h point for threshold. Most used a manual counting method (Makler chamber, out of a minimum of 100 or 200 spermatozoa), whereas one company used automated counting. Others did not detail their counting techniques. HSSA objectives and thresholds The HSSA objectives and thresholds varied from one company to another. Most of the companies used a cut-off of 70 or 75% SMI after 24 h of incubation. One manufacturer recorded motility after 2 and 24 h of incubation and expected SMI >75%. Another company had a SMI target of >72% after 8 h of incubation, and finally, for one company, the SMI should be >70% for the test and >75% for the control groups within 24, 48 and 72 h of incubation. Discussion This study shows significant differences in methodologies and objectives of MEA and HSSA used by manufacturers to confirm the absence of toxicity in their IVF consumables. As regards MEA testing, the mouse strain (outbred, inbred or hybrid), the number and the origin (fresh or thawed) of the embryos as well as their stage (one-cell or two-cell) differed from one manufacturer to another. Importantly, these parameters could influence the sensitivity of the tests (Davidson et al., 1988, Fleetham et al., 1993, Gardner et al., 2005, Hendriks et al., 2005, Khan et al., 2013, Kruger and Stander, 1984, Naz et al., 1986, Punt-van der Zalm et al., 2009, Van den Abbeel et al., 1999, Wolff et al., 2013, Zarmakoupis-Zavos and Zavos, 1996). In particular, the number of embryos used varied from 15 to 31 per group with differences in numbers between the test and the control groups. In this aspect Punt-van der Zalm et al. observed that a minimum of 69 embryos was necessary to detect the toxicity with an acceptable level of sensitivity (Punt-van der Zalm et al., 2009). In our survey, we were not able to disclose whether and how embryos were randomized although this point is important due to between-mouse and within-mouse variations (Hendriks et al., 2005, Khan et al., 2013). In the same way, the culture conditions (protein supplementation, embryo density, oil, hypoxia…) of the MEA differed among companies although they could impact the results (Ainsworth et al., 2017, Bontekoe et al., 2012, Gilbert et al., 2016, Otsuki et al., 2007, Otsuki et al., 2009). As an example, proteins like albumin could bias the results as they can chelate toxins (Fissore et al., 1989, Flood and Shirley, 1991, Silverman et al., 1987). Our observations regarding HSSA were not different from those of MEA with differences in terms of thresholds, temperatures and incubation times among companies. Moreover, the origin (fresh or thawed) of the donor sperm, sperm parameters (concentrations) and sperm preparation techniques as well as culture media were mostly unknown. However, all these parameters have been shown to influence the HSSA results (Claassens et al., 2000, Critchlow et al., 1989, De Jonge et al., 2003, Iemmolo et al., 2005, Lierman et al., 2007). Another weakness of the HSSA is that it was mostly performed with only one sperm sample per device, thereby being sensitive to inter-assay variation (Claassens et al., 2000). In future regulations, a statistically validated number of samples should be described. Fluctuations in supply quality in the IVF laboratory occurs despite bioassay testing by the supplier, showing that the current toxicity screening processes could be deficient (Morbeck, 2012). Indeed, Nijs et al. observed that 8% of the IVF devices were found to be potentially embryotoxic when re-tested with HSSA before use (Nijs et al., 2009). Moreover, reports of toxicity of devices used in ART laboratories all use binary tests (yes/no) with few samples tested and, with respect to plastics, the source of toxicity has never been defined. In contrast, toxins in mineral oil (Morbeck, 2012) and protein (Leonard et al., 2013) have been identified and relative toxicity of identified toxins have been compared in a dose-dependent manner with the most common assays used by manufacturers (Hughes et al., 2010). A limitation of the present study is the number of participating companies and a rather low response rate (11/21). However, during the study we tried our best to increase the response rate by repeated contacts to the companies, and it was clear that there was quite a high degree of unwillingness to answer the questionnaire which touched upon delicate matters for the companies. In contrast, the strength of the study is the fact that the main companies working in the field of IVF were contacted—and if they did not respond, several attempts were made to obtain contact. Although the response rate was low, we believe that the present results depict the current status of embryo toxicity testing with a lack of standardization and regulation and a very poor transparency. Importantly, our study confirms the heterogeneity of embryotoxicity testing performed by manufacturers when validating their IVF disposables. The ‘discipline’ of toxicity testing of devices and media used in the IVF laboratory is currently poorly developed, which leaves manufacturers and regulatory bodies with limited evidence to choose the ‘best practice’. The question of sensitivity of testing was previously underlined (Tucker and Jansen, 2002, Wetzels et al., 2010), and recommendations and standardization are obviously needed to improve the sensitivity and reproducibility over time. Some companies, conscious of the weakness of embryotoxicity testing, have developed alternative tests based on novel technologies to improve sensitivity (Ainsworth et al., 2017, Gilbert et al., 2016, Mestres et al., 2019, Wolff et al., 2013), and US FDA and UE concept guidelines are in progress on this subject (Mouse Embryo Assay for Assisted Reproduction Technology Devices, Draft guidance for industry and food and drug administration staff). Otherwise, the information provided by manufacturers on this important subject is quite poor, and it is clear from our analysis that there is an urgent need for regulation and transparency within the area of embryo toxicity testing of IVF disposables. This problem of transparency also occurs for commercial culture media, the composition of which is not fully disclosed to users although it seems that different brands lead to differences in birth weight (Kleijkers et al., 2016) and child’s weight (Zandstra et al., 2018). In conclusion, significant differences in methodology and objectives of toxicity testing of IVF disposables were seen in this questionnaire-based study; although the response rate was rather low, the results are likely to depict the current status. In this context and waiting for regulatory improvements on toxicity testing, we recommend that IVF laboratories’ re-test critical products prior to use as a part of their quality management programs. Acknowledgements The authors thank Dr Etienne Ruppé for his careful reviewing of the manuscript. The authors thank the following for their responses to the questionnaire: Jeff Lascher (Cook Medical), Magdalena Depa-Martinow (Cook Medical), Nadia Touamine (Cook Medical), Isabelle Lorenzatto (Vitrolife), Hubert Joris (Vitrolife), Sophie Jolly (CooperSurgical), Xavier Buchon (CooperSurgical), Mylène Santos (CooperSurgical), Isabelle Calais (CooperSurgical), Patrick Choay (CCD), Patrick Bouveret (CCD), Mohamed Bidri (CCD), Florence Vullierme (CCD), Bernard Montanari (ElliosBioTek), Catherine Broberg (E.in.Art), Will Veen (WillcoWells), Marie Josée Ze-Bissemou (ThermoFischer Scientific), Raquel Matos (Corning Life Science), Vincent Yang (Sunlight Medical), Julio Piedra (Kitazato Dibimed), Yannick Feirrera (Merck Serono), Becca Swan (Gynetics), Michelle Tedder (EmbryoTech), Melissa Alfaro (EmbryoTech), Layla El Younani (JCD), Jean Louis Prevost (JCD), Daniel Schnitzer (Biocare), Jean Louis Spach (Biocare), Cécile Sudon (Biocare), Alice de Ligniville (Charles River) and Benoit Remy (GIGA). Authors’ roles L.D. designed the study, did the data analysis and wrote the article. C.D. and P.H. revised the article and approved the final draft. All of the authors contributed to the scientific discussions and reviewed the article. Funding No funding was received for this study. Conflict of interest L.D. declares a competing interest with Patrick Choay SAS. References Ackerman S , Swanson R , Stokes G , Veeck L . Culture of mouse preimplantation embryos as a quality control assay for human in vitro fertilization . Gamete Res 1984 ; 9 : 145 – 152 . Google Scholar Crossref Search ADS WorldCat Ackerman SB , Stokes GL , Swanson RJ , Taylor SP , Fenwick L . Toxicity testing for human in vitro fertilization programs . J In Vitro Fert Embryo Transf 1985 ; 2 : 132 – 137 . Google Scholar Crossref Search ADS PubMed WorldCat Ainsworth AJ , Fredrickson JR , Morbeck DE . Improved detection of mineral oil toxicity using an extended mouse embryo assay . 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Clinical Evaluation: a guide for manufacturers and notified bodies under directives 93/42/EEC and 90/385/EEC. June . In: Guidelines on Medical Devices , 2016 Medical Device Directive MDD: Council Directive 93/42/EEC . of 14 June 1993 concerning medical devices Official Journal of 12.07.1993, L169 . Available from : https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:31993L0042. Mestres E , Garcia-Jimenez M , Faes L , Vanrell I , Bogaert V , Jonckheere I , Casals A , Llop C , Senti M , Calderon G et al. Parameters of the mouse embryo assay that affect detection of peroxides in mineral oil . Reprod Biomed Online 2019 ; 39 : 547 – 555 . Google Scholar Crossref Search ADS PubMed WorldCat Morbeck DE . Importance of supply integrity for in vitro fertilization and embryo culture . Semin Reprod Med 2012 ; 30 : 182 – 190 . Google Scholar Crossref Search ADS PubMed WorldCat Naz RK , Janousek JT , Moody T , Stillman RJ . 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Association of culture medium with growth, weight and cardiovascular development of IVF children at the age of 9 years . Hum Reprod 2018 ; 33 : 1645 – 1656 . Google Scholar Crossref Search ADS PubMed WorldCat Zarmakoupis-Zavos PN , Zavos PM . Factors that may influence the mouse embryo bioassay . Tohoku J Exp Med 1996 ; 179 : 141 – 149 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Embryotoxicity testing of IVF disposables: how do manufacturers test?

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Oxford University Press
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© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.
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Abstract

Abstract STUDY QUESTION How do manufacturers perform embryotoxicity testing in their quality control programs when validating IVF consumables? SUMMARY ANSWER The Mouse Embryo Assay (MEA) and Human Sperm Survival Assay (HSSA) used for IVF disposables differed from one manufacturer to another. WHAT IS KNOWN ALREADY Many components used in IVF laboratories, such as culture media and disposable consumables, may negatively impact human embryonic development. STUDY DESIGN, SIZE, DURATION Through a questionnaire-based survey, the main manufacturers of IVF disposable devices were contacted during the period November to December 2018 to compare the methodology of the MEA and HSSA. We focused on catheters for embryo transfer, catheters for insemination, straws, serological pipettes, culture dishes and puncture needles used in the ART procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS We approached the manufacturers of IVF disposables and asked for details about methodology of the MEA and HSSA performed for toxicity testing of their IVF disposable devices. All specific parameters like mouse strains, number of embryos used, culture conditions (media, temperature, atmosphere), extraction protocol, subcontracting, and thresholds were registered and compared between companies. MAIN RESULTS AND THE ROLE OF CHANCE Twenty-one companies were approached, of which only 11 answered the questionnaire. Significant differences existed in the methodologies and thresholds of the MEA and HSSA used for toxicity testing of IVF disposables. Importantly, some of these parameters could influence the sensitivity of the tests. LIMITATIONS, REASONS FOR CAUTION Although we approached the main IVF manufacturers, the response rate was relatively low. WIDER IMPLICATIONS OF THE FINDINGS Our study confirms the high degree of heterogeneity of the embryotoxicity tests performed by manufacturers when validating their IVF disposable devices. Currently, no regulations exist on this issue. Professionals should call for and request standardization and a future higher degree of transparency as regards embryotoxicity testing from supplying companies; moreover, companies should be urged to provide the users clear and precise information about the results of their tests and how testing was performed. Future recommendations are urgently awaited to improve the sensitivity and reproducibility of embryotoxicity assays over time. STUDY FUNDING/COMPETING INTEREST(S) This study did not receive any funding. L.D. declares a competing interest with Patrick Choay SAS. TRIAL REGISTRATION NUMBER N/A embryotoxicity testing, human sperm survival assay, mouse embryo assay, IVF disposable, sensitivity Introduction The success of IVF depends on the quality of laboratory procedures, environmental conditions, equipment and reagents, which should be monitored and improved through quality control testing (Gardner et al., 2005, Lane et al., 2008, Morbeck, 2012). Toxicity of supplies used for embryo culture has been well documented and recent reports provide evidence that the quality of supplies is variable (Lierman et al., 2007, Morbeck, 2012, Nijs et al., 2009). Recommendations advocate testing media and consumables before their use due to the potential release of toxic substances which might affect gametes and embryos (Lane et al., 2008, Morbeck, 2012). Indeed, the ubiquitous use of IVF disposable plastics makes them one of the providers of embryotoxic substances (Cohen et al., 1997, DeHaan, 1971, Holyoak et al., 1996, Nijs et al., 2009). Plastics are not usually toxic; however, changes may occur during sterilization, packaging and storage, resulting in the release of breakdown products, such as short hydrocarbon chains, volatile organic compounds (VOC), and in modifications of the electrical charge on their surfaces (Lee, 1999, Sladowski et al., 2008, Vardhan and Shukla, 2018). The toxicity of plastics may also derive from additives (anti-static or anti-viscous agents) which facilitate the transformation of resins and increase their stability (Olivieri et al., 2012). Importantly, embryotoxicity could lead to immediate apoptosis of the gametes and embryos, even after brief contact with the plastic disposable (Nijs et al., 2009, Ray et al., 1987). Thus, embryotoxicity should be suspected in case of decreased fertilization rates or abnormal embryonic development (Nijs et al., 2009), and long-term health consequences in the offspring cannot be ruled out (Morbeck, 2012). Differences in birthweight (Banrezes et al., 2011), imprinting (Fernandez-Gonzalez et al., 2004, Lonergan et al., 2003, Rivera et al., 2008) and gene expression pattern (Ecker et al., 2004) in domestic animal species have been observed when embryos were cultured under sub-optimal conditions. In humans, evidence is accumulating that in vitro culture conditions may influence the perinatal outcomes (Marianowski et al., 2016, Sunde et al., 2016, Zandstra et al., 2018). Moreover, embryotoxicity is dose-cumulative, in the sense that a component with minimal intrinsic toxicity could see its toxicity amplified when combined with other stress factors (Awonuga et al., 2013, Wale and Gardner, 2016). Faced with the in vitro chemical threats, the embryo uses different defence mechanisms, requiring large amounts of energy which hampers development. In this aspect, it is, therefore, essential to check the possible toxicity of IVF consumables before implemented into IVF procedures. Currently, two main bioassays are used to assess the safety of reagents and materials used for IVF. The main test is the mouse embryo assay (MEA) (Ackerman et al., 1984, Ackerman et al., 1985, Fleetham et al., 1993, Parinaud et al., 1987) which involves culturing one-cell or two-cell mouse embryos (Ackerman et al., 1985) up to the blastocyst stage in a culture medium, previously in contact with the consumable being tested. The subsequent blastulation rate is used as a parameter of possible toxicity. A second bioassay is the human sperm survival assay (HSSA) (Bavister and Andrews, 1988, Claassens et al., 2000, Critchlow et al., 1989). This test measures the survival (or the motility) of human sperm after the exposure to the consumable, comparing it to those of non-exposed sperms at specific observation times. The result is expressed as the sperm motility index (SMI) which is calculated by the ratio of the sperm motility of the sample tested divided by the motility of the control. Currently no toxicity testing regulations exist for manufacturers of IVF disposables—only guidelines. In the USA, the current good manufacturing practice of the US Food and Drug Administration (FDA) requires that toxicity tests like MEA should be carried out in culture media and consumables used for IVF without the methodology being specified (FDA, 1998). Similarly, in the EU, manufacturers must demonstrate safety and effectiveness of their products to obtain a CE mark (Medical Device Directive MDD, 1993, MEDDEV 2.7/1 revision 4); however, the mode is not detailed. Thus, there are no well-defined safety and quality standards for ART devices, focussing on safety of gametes, embryos, and offspring (Wetzels et al., 2010). The aim of the present study was to explore the methodology employed for MEA and HSSA testing in quality control programs by the main manufacturers of IVF consumables. Materials and Methods Study design Between November and December 2018, a questionnaire was forwarded to the main manufacturers and distributors of consumables used for IVF in order to determine the methodology used for MEA and HSSA. The questionnaire was also sent to three subcontractors. The products studied were catheters for embryo transfer, catheters for insemination, straws, serological pipettes, culture dishes and puncture needles used in the IVF procedures. Each company was asked to answer the questionnaire (one answer per type of device). In case of no-reply, companies were approached either by telephone or by repeated mail in which case up to five reminder messages were forwarded. Questionnaire Thirty-four questions, 21 on the MEA and 13 on the HSSA, were included in the questionnaire. The following data were recorded for the MEA: mouse strain, number of embryos used for testing, type of embryo (one- or two-cell), origin of the embryos (fresh or frozen), culture conditions (culture medium, micro-drops or wells, individual or grouped culture, %O2, temperature, air quality). Manufacturers were asked about possible subcontracting, the type of products used for testing (raw materials or final products), the number of items tested per lot and the certification of their quality control programs. Moreover, questions included the extraction protocols, the objectives and the accepted toxicity thresholds. Finally, manufacturers were asked if they performed a cell count of the trophectoderm and intra-cellular cell mass (ICM) of blastocysts obtained from the MEA (methodology, number of studied blastocysts, objectives and thresholds). For the HSSA, manufacturers were questioned about subcontracting, air quality during testing, categories and number of tested devices per lot, objectives and the accepted toxicity thresholds. Moreover, the following data were recorded: the extraction protocol, the type of counting technique (manual or automated), the origin of the sperm sample (fresh or frozen), the sperm sample concentration, the type of sperm selection (gradient, swim-up, washing), the medium used, the incubation temperature, the number of sperms counted and the type of motility evaluated (total and progressive). Results In total, 21 companies were approached. Eleven companies answered the questionnaire. Two companies declined to answer due to ‘confidentiality problems’. Three companies replied that they were only distributors and that the manufacturer should be approached directly. Five companies never replied despite five attempts to reach them via either phone or mail. One of three subcontractors answered the questionnaire, one declined to participate and the last subcontractor did not reply despite five reminder messages. MEA The findings of the MEA are summarised in Table I. Among the 11 companies who answered the questionnaire, nine subcontracted their MEA. However, the methodology and the objectives used were not necessarily similar. One company would subcontract, depending on the geographic location of its factories. Another did not subcontract at all. When subcontracted, the tests met the International Organization for Standardization (ISO) 17025 standard and Good Laboratory Practice for Nonclinical Laboratory Studies, Title 21 Code of Federal regulations (CFR) Part 58 Accreditation Program for testing. MEA questions and answers. Table I MEA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) MEA: Mouse Embryo Assay ISO: International Organization for Standardization HTF®: HTF® (Human Tubal Fluid) Medium (Biocare, Rome, Italy) Global®: Global® (LifeGlobal Group LCC, Guilford, USA) BSA: bovine serum albumin EmbryoMax® Modified M16®: EmbryoMax® Modified M16 Medium (Sigma-Aldrich, Saint Louis, USA) EmbryoMax® KSOM®: EmbryoMax® KSOM (potassium-supplemented simplex optimised medium) Medium (Sigma-Aldrich, Saint Louis, USA) Open in new tab Table I MEA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 9) No (n = 1) Sometimes (n = 1) Do you test raw materials or only finished products? Raw materials and finished products (n = 2) Finished products only (n = 9) How many items do you test per lot? 10 items every 3 lots (n = 1) A minimum of 3 up to 10 items, depending on the lot size (n = 1) According to internal specifications compliant with ISO standards on sampling requirements (n = 1) 2 test articles. The second (retained test item) is for use in a retest (n = 4) 2 test articles. The second (retained test item) is for use in a retest. In total 10 items (1 box of products) (n = 1) 4 or 10 (n = 2) Minimum 2, mostly 4–5 devices are tested (n = 1) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) Which mouse strain do you use? B6C3F1xB6D2F1 (n = 6) B6/CBA (n = 1) FVB/NxCD1 (n = 1) Two strains of mice used. Type of mouse strain dependent on test specifications/requirements (n = 1) Multiple, depending on device and lab testing (n = 1) No answer (n = 1) How many embryos do you test? 15 embryos for control assay and 21 embryos for test assay (n = 7) 21 embryos for control assay and 21 embryos for test assay (n = 2) 30 embryos for control assay and 30 embryos for test assay (n = 1) 31 embryos for control assay and 31 embryos for test assay (n = 1) Do you use 1-cell or 2-cell embryos? 1 cell (n = 8) 2 cell (n = 3) Do you use fresh or frozen embryos? Fresh (n = 3) Typically, fresh, thawed used if necessary (n = 2) Randomly from a common pool of freshly collected embryos (n = 5) No answer (n = 1) Which culture medium do you use? Medium for human IVF (i.e. HTF®, Global® supplemented with 0.4% BSA) (n = 8) Medium for murine IVF (i.e. EmbryoMax® Modified M16®, EmbryoMax® KSOM®) (n = 2) For raw material, specially designed mouse medium stressing development. For final product, test-dependent (n = 1) Do you use micro-drops or wells? Typically, 1 mL droplets (n = 1) Microdrops (n = 3) Triplicate microdrops (three 12.5 μL microdrops) (n = 5) Microdrops 50 μL (n = 1) No answer (n = 1) How many embryos per micro-drop or per well? 7 per drop for the test item and 5 per well for the control (n = 5) Test-dependent (n = 1) Between 2 and 3 (n = 1) 21 per microdrop (n = 1) No answer (n = 3) Which temperature? 37 ± 1°C (n = 11) 20% O2 or 5% O2? 20% O2 (n = 11) Cleanroom-like controlled environment? Yes (n = 1) No (n = 2) No answer (n = 8) Objectives/thresholds? ≥80% blastocyst development within 96 h (n = 1) ≥75% blastocyst development within 96 h (n = 1) ≥80% expanded blastocyst development within 96 h (n = 4) ≥80% of 2 cell control embryos to develop to expanded blastocysts within 72 h (n = 1) ≥70% expanded blastocyst development within 96 h (n = 1) ≥80% of embryos must proceed from 1 to 2 cells in 24 h and at least 80% of embryos must proceed to expanded blastocyst stage within 96 h (n = 1) ≥80% of 1 cell stage embryos to develop to 2 stage within 24 h and hatching blastocysts within 120 h (n = 1) Timely development at fixed time points (Days 2, 3 and 4) with >80% blastulation and cell number not different from control (n = 1) Certification ISO 17025:2017 of the quality controls? Yes (n = 9) No answer (n = 2) Do you perform blastocyst cell counting after MEA? Yes (n = 3) No (n = 8) If yes, for which devices? All (n = 1) If requested (n = 1) No answer (n = 1) Methodology? Fluorescence microscopy (n = 3) Objectives/thresholds? Not different from control (n = 3) How many blastocysts? All blastocysts (n = 3) MEA: Mouse Embryo Assay ISO: International Organization for Standardization HTF®: HTF® (Human Tubal Fluid) Medium (Biocare, Rome, Italy) Global®: Global® (LifeGlobal Group LCC, Guilford, USA) BSA: bovine serum albumin EmbryoMax® Modified M16®: EmbryoMax® Modified M16 Medium (Sigma-Aldrich, Saint Louis, USA) EmbryoMax® KSOM®: EmbryoMax® KSOM (potassium-supplemented simplex optimised medium) Medium (Sigma-Aldrich, Saint Louis, USA) Open in new tab MEA testing Two companies performed MEA on both raw materials and final products, nine companies on the finished products, only. MEA was performed on packed and sterilized products without pre-stage ‘off gassing’ except for one company which opened packages and let them air overnight prior to MEA testing. The number of items tested per lot varied according to the company and the type of consumable, from 1 to 10 per lot, with one company performing MEA on 10 items every 3 lots. Six manufacturers performed MEA tests using hybrids from inbred strains B6C3F1xB6D2F1. One company used strain B6/CBA and another used strain FVB/NxCD1. For two other companies, several different strains of mice were used according to the item tested and the specificity of the requests. A minimum of 15 embryos were used, sometimes as many as 30 or 31. All companies performed MEA with a control group, but the number of embryos used could differ between the test group and control group. Randomization of the embryos was not performed by all. Eight companies used one-cell embryos, and three companies used two-cell embryos. Most embryos were fresh or occasionally a pool of fresh and frozen embryos. Culture media and culture Eight companies used a culture medium for human IVF and two companies used a culture medium for murine IVF. The majority of MEA tests were performed in batched culture. Two, 3, 5, 7 or 21 embryos were dispensed per micro-drop or well, the volume of medium varied from 12.5 μL to 1 mL. Culture was usually carried out at 37 ± 1°C in 5 ± 1% CO2. No test was carried out under hypoxic conditions. The air quality of the laboratory in which the tests were carried out was monitored by one company, only. Two companies answered that the air quality was not monitored; the other eight companies chose to not answer this question. The extraction protocol used for MEA varied according to the manufacturer and the type of devices tested. Examples of extraction protocols on embryo transfer catheters and IVF dishes are presented in Table II. Examples of extraction protocols used by the different companies for MEA or HSSA. Table II Examples of extraction protocols used by the different companies for MEA or HSSA. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. MEA: Mouse Embryo Assay HSSA: Human Sperm Survival Assay Open in new tab Table II Examples of extraction protocols used by the different companies for MEA or HSSA. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. COMPANY 1 COMPANY 2 COMPANY 3 MEA on embryo transfer catheters Attach a sterile syringe to the catheter hub and flush culture medium through the syringe 3 times for 30 s, expelling into a Falcon tube. Cut the tip of the catheter approximately 4 cm from the end and soak it in culture medium in the Falcon tube for 2 h at room temperature. 3 mL of culture medium are flushed through the liquid path of the test item at 25°C for 60 s (medium is discarded after flush). Post flushing, 1 mL of culture medium (25°C, 20 s) is used to culture 1-cell mouse embryos for 96 h. The extracts are transferred to culture plates (60-well) overlaid with oil and equilibrated in a CO2 incubator containing 5% CO2 (± 1%) and 37°C (± 1°C) for at least 1 h before receiving the embryos to assure correct temperature and pH. Positive control is set up in the same culture plate as the test item. 0.5 or 2 mL (depending on the catheter size) of culture medium are flushed through the liquid path of the test item at 25°C for 30 s. This medium is used to incubate 2-cell embryos for 30 min. Embryos are then transferred to M16® medium for 72 h. MEA on IVF dishes 1 mL of culture medium is placed in the test item and overlaid with oil. One-cell mouse embryos are placed in the 1 mL drop of culture medium and cultured for 96 h. All test and control embryos are selected randomly from a common pool of freshly collected embryos and are cultured in the same incubator at 37°C and 5% CO2. The central area of IVF dishes is prepared with tested culture medium microdroplets, immediately covered with oil and equilibrated overnight. Fresh 1-cell mouse embryos, collected from F1 hybrid females (B6/CBA) crossed with males from the same genetic background, are washed thoroughly and cultured in the IVF dishes for 3 h at 37.3°C and optimal %CO2 and %O2. Embryos are then transferred to previously tested culture dishes (prepared in triplicate and pre-equilibrated overnight) and cultured for 5 days. Control group is prepared in same way, and embryos cultured in parallel using test medium not exposed to samples. Embryo development of test and control groups are followed every 24 h and photos of final stages are taken. 1 mL of control medium is placed in the centre well of 3 test articles and incubated for 1 h. Post 1 h incubation, the medium is extracted from each of the 3 test articles and pooled. 1 mL of the pooled extracted medium is placed into the 4th test article, overlaid with oil and equilibrated for 1 h prior to receiving 1-cell mouse embryos. HSSA on insemination catheters The test article was flushed with 1 mL of sperm medium and incubated for 30 minutes. Post incubation, the test article was removed from the sperm medium and the prepared human sperm was added to the flushed sperm medium and incubated for 24 h at 32°C under 5% CO2. Direct testing: A concentration of sperm was drawn into the test article and placed in an incubator at 32°C in 5%CO2 for 24 h after which the sperm was expelled from the test article and examined for forward progressive motility. A human sperm sample was processed in duplicate, using silica-based density gradients (95%, 70% and 45% concentration) and test product incubated with previously tested medium for 60 min at 37°C. Afterwards, the resulting sperm preparation was incubated with the extracted medium (test group) or control tested medium (control group). Test and control groups were incubated in parallel in the same incubator chamber at 37.3°C and optimal % CO2 and % O2, and sperm motility was assessed at 0, 4, 8 and 24 h after processing. MEA: Mouse Embryo Assay HSSA: Human Sperm Survival Assay Open in new tab MEA objectives The MEA objectives were different among companies (Table I). Three companies used a cut-off of 75% or 80% blastulation rate within 96 h. Six companies added morphological criteria and used a cut-off of 70% or 80% expanded blastocysts rate within 72 or 96 h of culture. Two companies used a cut-off of 80% cleavage rate within 24 h associated to a cut-off of an 80% expanded blastocyst rate within 96 h, or a cut-off of an 80% hatching blastocyst rate within 120 h of culture. Moreover, some companies recorded differences in morphology between the test and control groups. Three companies carried out cell counting of the ICM and trophectoderm by fluorescent microscopy for all blastocysts obtained from the MEA, and the number of cells obtained per blastocyst should not be significantly different from that of the control group. HSSA The findings of the HSSA are summarised in Table III. In contrast to the MEA, the HSSA was not carried out systematically in all consumables used for IVF, but only on items that came into direct contact with sperms, such as insemination catheters, serological pipettes, and some of culture dishes. One manufacturer did not perform the HSSA due to lack of standardization and certification of the test. Six companies subcontracted the test. However, the methodologies and objectives used were not necessarily similar. Usually, a single item was tested per lot, with re-testing performed on a second article if the first test was negative. HSSA questions and answers. Table III HSSA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) HSSA: Human Sperm Survival Assay SMI: sperm motility index Open in new tab Table III HSSA questions and answers. Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) Questions Answers (1 by manufacturer) Do you subcontract? Yes (n = 6) No (n = 1) Do you use a cleanroom-like controlled environment? Yes (n = 1) No (n = 1) No answer (n = 5) Do you use different methodology according to the item? Yes (n = 2) No (n = 5) No answer (n = 4) How many items do you test per lot? According to international specifications compliant with ISO standard on sampling requirements (n = 1) 2 items. The second is for retest (n = 3) No answer (n = 3) Do you use fresh or frozen donor sperm? Fresh (n = 5) No answer (n = 2) Sperm concentrations? Sample variation (n = 1) 4–7 million/mL (n = 1) No answer (n = 5) Which sperm preparation? Gradient (n = 3) Swim-up (n = 1) No answer (n = 3) Which temperature of incubation? 37°C (n = 2) 32°C (n = 3) 32°C under 5% CO2 (n = 2) Which medium? With or without protein? Test-dependent (n = 1) HTF® (n = 1) Sperm washed with human albumin (n = 1) No answer (n = 4) Which motility? Progressive or total? Both (n = 2) Total probably (n = 1) Progressive (n = 3) No answer (n = 1) Do you use an automated method like CASA? Yes (n = 1) No (n = 2) How many sperms counted for motility? 200 (n = 2) 100 (n = 1) No answer (n = 4) Objectives/thresholds? Test-dependent (n = 1) SMI >70% at 24 h (n = 2) SMI >75% at 24 h (n = 1) SMI >75% at 2 h and 24 h (n = 1) SMI >75% and controls >70% at 24, 48 and 72 h (n = 1) SMI >72% by 8 h (n = 1) HSSA: Human Sperm Survival Assay SMI: sperm motility index Open in new tab Sperm and incubation Mostly fresh donor sperm was used; however, the initial and final concentrations of sperm used were rarely described. Likewise, the sperm preparation protocol was rarely described, usually being either gradient or swim-up. The incubation temperature used was 32 or 37°C, and sperm preparations were sometimes incubated under 5% CO2 in different types of media. As for MEA, the extraction protocol used for HSSA was different among companies. An example of insemination catheters is provided in Table II. Some manufacturers evaluated the total motility, others the progressive motility or both, mostly after 24 h of incubation. One company measured motility after 48 and 72 h incubation. Another company recorded motility at 0, 4, 8 and 24 h but used the 8-h point for threshold. Most used a manual counting method (Makler chamber, out of a minimum of 100 or 200 spermatozoa), whereas one company used automated counting. Others did not detail their counting techniques. HSSA objectives and thresholds The HSSA objectives and thresholds varied from one company to another. Most of the companies used a cut-off of 70 or 75% SMI after 24 h of incubation. One manufacturer recorded motility after 2 and 24 h of incubation and expected SMI >75%. Another company had a SMI target of >72% after 8 h of incubation, and finally, for one company, the SMI should be >70% for the test and >75% for the control groups within 24, 48 and 72 h of incubation. Discussion This study shows significant differences in methodologies and objectives of MEA and HSSA used by manufacturers to confirm the absence of toxicity in their IVF consumables. As regards MEA testing, the mouse strain (outbred, inbred or hybrid), the number and the origin (fresh or thawed) of the embryos as well as their stage (one-cell or two-cell) differed from one manufacturer to another. Importantly, these parameters could influence the sensitivity of the tests (Davidson et al., 1988, Fleetham et al., 1993, Gardner et al., 2005, Hendriks et al., 2005, Khan et al., 2013, Kruger and Stander, 1984, Naz et al., 1986, Punt-van der Zalm et al., 2009, Van den Abbeel et al., 1999, Wolff et al., 2013, Zarmakoupis-Zavos and Zavos, 1996). In particular, the number of embryos used varied from 15 to 31 per group with differences in numbers between the test and the control groups. In this aspect Punt-van der Zalm et al. observed that a minimum of 69 embryos was necessary to detect the toxicity with an acceptable level of sensitivity (Punt-van der Zalm et al., 2009). In our survey, we were not able to disclose whether and how embryos were randomized although this point is important due to between-mouse and within-mouse variations (Hendriks et al., 2005, Khan et al., 2013). In the same way, the culture conditions (protein supplementation, embryo density, oil, hypoxia…) of the MEA differed among companies although they could impact the results (Ainsworth et al., 2017, Bontekoe et al., 2012, Gilbert et al., 2016, Otsuki et al., 2007, Otsuki et al., 2009). As an example, proteins like albumin could bias the results as they can chelate toxins (Fissore et al., 1989, Flood and Shirley, 1991, Silverman et al., 1987). Our observations regarding HSSA were not different from those of MEA with differences in terms of thresholds, temperatures and incubation times among companies. Moreover, the origin (fresh or thawed) of the donor sperm, sperm parameters (concentrations) and sperm preparation techniques as well as culture media were mostly unknown. However, all these parameters have been shown to influence the HSSA results (Claassens et al., 2000, Critchlow et al., 1989, De Jonge et al., 2003, Iemmolo et al., 2005, Lierman et al., 2007). Another weakness of the HSSA is that it was mostly performed with only one sperm sample per device, thereby being sensitive to inter-assay variation (Claassens et al., 2000). In future regulations, a statistically validated number of samples should be described. Fluctuations in supply quality in the IVF laboratory occurs despite bioassay testing by the supplier, showing that the current toxicity screening processes could be deficient (Morbeck, 2012). Indeed, Nijs et al. observed that 8% of the IVF devices were found to be potentially embryotoxic when re-tested with HSSA before use (Nijs et al., 2009). Moreover, reports of toxicity of devices used in ART laboratories all use binary tests (yes/no) with few samples tested and, with respect to plastics, the source of toxicity has never been defined. In contrast, toxins in mineral oil (Morbeck, 2012) and protein (Leonard et al., 2013) have been identified and relative toxicity of identified toxins have been compared in a dose-dependent manner with the most common assays used by manufacturers (Hughes et al., 2010). A limitation of the present study is the number of participating companies and a rather low response rate (11/21). However, during the study we tried our best to increase the response rate by repeated contacts to the companies, and it was clear that there was quite a high degree of unwillingness to answer the questionnaire which touched upon delicate matters for the companies. In contrast, the strength of the study is the fact that the main companies working in the field of IVF were contacted—and if they did not respond, several attempts were made to obtain contact. Although the response rate was low, we believe that the present results depict the current status of embryo toxicity testing with a lack of standardization and regulation and a very poor transparency. Importantly, our study confirms the heterogeneity of embryotoxicity testing performed by manufacturers when validating their IVF disposables. The ‘discipline’ of toxicity testing of devices and media used in the IVF laboratory is currently poorly developed, which leaves manufacturers and regulatory bodies with limited evidence to choose the ‘best practice’. The question of sensitivity of testing was previously underlined (Tucker and Jansen, 2002, Wetzels et al., 2010), and recommendations and standardization are obviously needed to improve the sensitivity and reproducibility over time. Some companies, conscious of the weakness of embryotoxicity testing, have developed alternative tests based on novel technologies to improve sensitivity (Ainsworth et al., 2017, Gilbert et al., 2016, Mestres et al., 2019, Wolff et al., 2013), and US FDA and UE concept guidelines are in progress on this subject (Mouse Embryo Assay for Assisted Reproduction Technology Devices, Draft guidance for industry and food and drug administration staff). Otherwise, the information provided by manufacturers on this important subject is quite poor, and it is clear from our analysis that there is an urgent need for regulation and transparency within the area of embryo toxicity testing of IVF disposables. This problem of transparency also occurs for commercial culture media, the composition of which is not fully disclosed to users although it seems that different brands lead to differences in birth weight (Kleijkers et al., 2016) and child’s weight (Zandstra et al., 2018). In conclusion, significant differences in methodology and objectives of toxicity testing of IVF disposables were seen in this questionnaire-based study; although the response rate was rather low, the results are likely to depict the current status. In this context and waiting for regulatory improvements on toxicity testing, we recommend that IVF laboratories’ re-test critical products prior to use as a part of their quality management programs. Acknowledgements The authors thank Dr Etienne Ruppé for his careful reviewing of the manuscript. The authors thank the following for their responses to the questionnaire: Jeff Lascher (Cook Medical), Magdalena Depa-Martinow (Cook Medical), Nadia Touamine (Cook Medical), Isabelle Lorenzatto (Vitrolife), Hubert Joris (Vitrolife), Sophie Jolly (CooperSurgical), Xavier Buchon (CooperSurgical), Mylène Santos (CooperSurgical), Isabelle Calais (CooperSurgical), Patrick Choay (CCD), Patrick Bouveret (CCD), Mohamed Bidri (CCD), Florence Vullierme (CCD), Bernard Montanari (ElliosBioTek), Catherine Broberg (E.in.Art), Will Veen (WillcoWells), Marie Josée Ze-Bissemou (ThermoFischer Scientific), Raquel Matos (Corning Life Science), Vincent Yang (Sunlight Medical), Julio Piedra (Kitazato Dibimed), Yannick Feirrera (Merck Serono), Becca Swan (Gynetics), Michelle Tedder (EmbryoTech), Melissa Alfaro (EmbryoTech), Layla El Younani (JCD), Jean Louis Prevost (JCD), Daniel Schnitzer (Biocare), Jean Louis Spach (Biocare), Cécile Sudon (Biocare), Alice de Ligniville (Charles River) and Benoit Remy (GIGA). 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Journal

Human ReproductionOxford University Press

Published: Feb 29, 2020

References

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