fertility magazine

THE FIRST MAGAZINE IN FERTILITYTM Volume 19

MAGAZINE

ESHRE 2016, Helsinki

AIR QUALITY AND ITS IMPACT ON EMBRYO DEVELOPMENT: CONTROL OF AIR POLLUTION IN AN ART LABORATORY AND ADJACENT AREASby Toni Di Berardino, BSc, MSc

TROPHECTODERM BIOPSY: THE PULLING AND FLICKING TECHNIQUESby Nuno Costa-Borges, PhD and Gloria Calderón, PhD

IVF DATA FLOW: FROM TRASH BINS TO CLIPBOARDS TO EMR TO SMART APPSby Stephen Fiser, Giles Tomkin and Jacques Cohen

HOW TO ASSURE A STABLE TEMPERATURE IN CULTURE MEDIUM MICRODROPLETS?by Enric Mestres, Ivette Vanrell, Maria Garcia, Nuno Costa-Borges, and Gloria Calderón

The global® Family – A Unified Approach to Embryo Culture

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Fertility Magazine • Volume 19 • www.FertMag.com – Page 1

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Fertility Magazine • Volume 19 • www.FertMag.com

ContentsFertility Magazine The First Magazine In FertilityTM

The Featured ArticleAir quality and its impact on embryo development: control of air pollution in an ART Laboratory and adjacent areas by Toni Di Berardino, BSc, MSc ........................................................................6

Instructions to ContributorsTo submit an Article, Abstract or Ad email us at:

[email protected]

Note: Articles and Abstracts must beaccompaniedby a photo of the author(s).

Featured On The CoverTrophectoderm Biopsy: The Pulling and Flicking Techniquesby Nuno Costa-Borges, PhD and Gloria Calderón, PhD ....................................14

IVF Data Flow: From Trash Bins to Clipboards to EMR to Smart Appsby Stephen Fiser, Giles Tomkin and Jacques Cohen ............................................24

How to assure a stable temperature in culture medium microdroplets?by Enric Mestres, Ivette Vanrell, Maria Garcia, Nuno Costa-Borges, and Gloria Calderón .................................................................................................32

Toni Di BerarDino, BSc, MSc

chaSe & Griffinn


Fertility Magazine and all its associates ©2016, All Rights Reserved. Covers, contents, images, ads in print or web form are copryight protected and reprinting or reproduction of any kind is expressly prohibited without the written permission of Fertility Magazine. Fertility Magazine does not knowingly accept false or misleading advertising, articles, opinions or editorial, nor does the publisher assume any responsibility for the consequences that occur should any such material appear, and assumes no responsibility for content, text, opinions or artwork of advertisements appearing in Fertility Magazine in print or web form. Some of the views expressed by contributors may not be the representative views of the publisher.

Articles22 Optimizing success in reproductive techniques: decreasing emotional distress by improving

personal care. by Ana Rita S. Coutinho, clinical embryologist certified (CFAS), MDV, PhD

28 The Slower Growing Embryo – Is it Suitable for Day 5 Embryo Transfer and Day 6 Vitrification? by Carole Lawrence

38 Conferences

ESHRE 2015 – Lisbon, PortugalJuly 2015

ASRM 2015 – Baltimore, MarylandOctober 2015

Editor in Chief: Monica Mezezi, MBAEditor(s): Don Rieger, PhDAssistant Editor: Michael CecchiDesign: Debrah GopsillEditorial Office: LifeGlobal Group, LLC 24 Norwich St. E. Guelph, ON, Canada N1H 2G6

T: 1-800-720-6375F: 1-519-826-6947Intl.: 001-519-826-5800editor@LifeGlobal.comwww.LifeGlobalGroup.comwww.IVFonline.comwww.FertMag.com


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THE FEATURED ARTICLE

Toni Di BerarDino, BSc, MSc

The earliest stages of preimplantation embryos and the success of assisted reproduction techniques are particularly sensitive to many laboratory

factors such as osmolality and pH of the culture media, temperature, and oxygen tension used during embryo culture. Laboratory environment and air quality are other important factors that have been shown to influence the success of assisted reproduction techniques. Initially, these were derived from anecdotal exchanges of information until the first peer-reviewed publication on such issues. Cohen et al., (1997) noted that there was evidence that chemical air contamination (CAC) may inhibit all stages of embryo development. They reported that during a temporary relocation of their research laboratory, the nearby construction had a lethal effect on their mouse embryo development. Moreover, it was reported that outside air was cleaner than filtered laboratory air and this was due to the accumulation of volatile organic compounds (VOCs) from outside air and laboratory products and equipment. VOCs such as benzene, toluene, cyclene and styrene were detected inside incubators and this was probably due to the items placed inside them, especially the plasticware (Gilligan et al., 1997). Compressed CO2 bottles were another source of VOC contamination (Schimmel et al., 1997).

Initially, the focus was placed on the filtration of airborne particulates using high-efficiency particulate arresting (HEPA) air filters. HEPA filtration is rated as being 99.97% effective in removal of particles that are 0.3 microns but not an effective barrier to embryotoxic VOCs (Gilligan 2010). The typical VOC particle is 1-10 nanometers in size and therefore smaller than the effective pore size of the HEPA filter. Following the publication by Cohen et al., (1998) the chemical air filtration focused on the removal of VOCs with solid phase filtration such as activated carbon and potassium permanganate (KMnO4). The identification of VOCs and its impact on the performance of the IVF laboratory led to the development of the Coda® incubation chamber air filter (Morbeck 2015). Also in the late 1990s,

Coda® Inline® Filters (LifeGlobal Group) for medical gasses were introduced to the market and have now become common accessories in most IVF laboratories. Coda® Inline® Filters use coconut shell-based activated charcoal which has a large internal surface area making it highly absorptive and therefore able to remove high molecular weight VOCs such as benzene, toluene, and xylene. Recent studies performed on LifeGlobal Coda® Inline® Filters and incubator filters by Alpha Environmental for research and quality assurance,

tested two challenges of organics: VOCs and aldehydes and the results showed a removal of 98.1% to 99.9% of VOC mixture and 98.1% to 99.9% of aldehydes.

Locke et al., (1990) identified the four most common air pollutants as (i) volatile organic compounds (VOCs), (ii) small inorganic molecules, (iii) substance derived from building materials, and (iv) polluting

compounds released by pesticides or aerosols. The sources of air contamination are many. Motor vehicle exhaust is a common contaminant of “fresh” air intakes for building air conditioning systems in urban areas (Cohen et al., 1997). Road surfaces and coal tar derivatives contain acrolein, which is toxic to mouse embryos in vitro (Hall et al., 1998). Fresh asphalt is one of the greatest sources of embryotoxic toluene.

Other important contaminants are released from adhesives, packaging, insulation, construction materials, and cleaning products. Brown et al., (1999) showed that particle boards and other wood-based panels release formaldehyde. PVC flooring material (Lundgren et al., 1999), carpet (De Bortoli et al., 1999) and paint (Sparks et al., 1999) represent a major source of VOCs, releasing numerous VOCs including aldehydes. In addition, common laboratory chemicals such as ethanol, methanol, and isopropyl alcohol are toxic. Chlorhexidine is known to be toxic to human sperm (Lawrence, 2007). Perfumes and cosmetics are

Air quality and its impact on embryo development: control of air pollution in an ART Laboratory and adjacent areasby Toni Di Berardino, BSc, MScDirector of Clinical & Technical SupportLifeGlobal® Group, Guelph, Ontario, Canada

You can contact Toni Di Berardino at [email protected]

…it was reported that outside air was cleaner than filtered laboratory air…



toxic to embryos in vitro (Johnson et al., 1993) and most IVF clinics have adopted a “fragrance-free” environment. Most embryologists now understand the importance of air quality and its association with embryo development and laboratories are now outfitted with HEPA, activated carbon filters, and positive pressure for air particulate control. VOCs can easily enter the oil overlay and the media. Once present in the media or oil, the VOCs are a permanent resident in the culture environment and thus a permanent threat to the embryos. Subtle levels of airborne VOCs and biological contaminants can be devastating to successful human embryogenesis and clinical outcomes.

Since the first publication, many scientific papers have investigated the relationship between laboratory air quality and embryo development. Both human and animal studies have suggested a correlation between poor laboratory environment and a decrease in implantation and pregnancy rates. Merton et al., (2007) reported a significant increase of pregnancy rates for both fresh and frozen embryos in one bovine breeding program with the use of a Coda® incubator air filter system. Although there was no effect of Coda® filtration on numbers or quality of blastocysts, there were significant effects on pregnancy rates. This would suggest that Coda® filtration removed VOCs are having a significant effect on the embryos.

Published data on human IVF also indicate that new or upgraded facilities with air-filtering systems are often reporting higher human IVF outcomes. Racowsky et al., (1999) observed the benefit of the Coda® by reporting a lower miscarriage rate when the laboratory was outfitted with a portable filtration system; the Coda® Tower and Coda® Unit attached to incubators. Lopez et al., (2004) reported a positive effect of Coda® on fertilization, implantation and pregnancy rates. Forman et al., (2004) showed that particulates counts were reduced over a 24 hour period when a Coda® Tower was placed in the IVF laboratory. There was also a decrease of these particulates when compared to IVF laboratory air quality when only HVAC was used and in comparison to the surrounding area such as the procedure room. Increases in nitrogen dioxide (NO2) concentration both at the patient’s address and at the IVF lab was significantly associated with a lower chance of pregnancy and live birth during all phases of an IVF cycle (Legro et al., 2010).

Heitmann et al., (2015) were able to compare outcomes as a result of a required move where the air handling environment was changed with the introduction of Coda® Tower filter units in both laboratory and operating theatre. Also, each gas bottle was outfitted with a Coda® Inline® Filter. Air quality testing showed an improved environment in their new site and implantation rate reflected the improvements. A 25% (P < 0.001) increase in implantation rate and a 19% (P <0.05) increase in live birth rate was reported.

Air filtration systems are now common in IVF

laboratories, which are tightly controlled environments. Numerous studies have shown better IVF outcomes following improvement in laboratory and surrounding air quality. Mayer et al., (1999) showed improved pregnancy and implantation rates after using a Coda® system. It is essential to set up an air filtration system that is efficient and affordable but most importantly it must be capable of filtering VOCs, chemically active compounds and be able to eliminate airborne pathogens (Khoudja et al., 2013). Munch et al., (2015) were able to review data from three periods of time where active carbon air filtration was present. A period of embryo culture carried out in the absence of carbon air filtration was a final period of time when the carbon filtration was reinstalled. They concluded that the absence of carbon air filtration was associated with poor fertilization, cleavage, and blastocyst conversion. Recognizing the importance of air quality and its effect on embryo development was also identified as a critical parameter in top performing laboratories in the USA (Van Voorhis et al., 2010).

Accumulated evidence indicates that the laboratory and surrounding areas’ air quality play an important role in IVF treatments and their outcomes. This conclusion has led to an extensive framework of standards for IVF laboratories. Regulatory directives in The European Union and Brazil require strict and specific air quality control within the IVF laboratories. Under the Directive of the European Union, (European Union, 2004) there are specifications for particulates and micro-organism contamination however VOCs are ignored. It is well established that VOCs can have significant adverse effects on embryos in culture, however the Directive has not addressed how this source of contamination can negatively impact treatment efficacy (Mortimer 2005). In contrast, the Brazilian regulatory directive does include the use of carbon activated filters but the specifics are lacking (Esteves et al., 2013). The standards for the American laboratories are published by the American Society for Reproductive Medicine (ASRM) however the specifics for air quality arer not addressed (ASRM, 2008).

In conclusion, since the first peer-reviewed paper by Cohen et al., (1997) an increasing amount of evidence is available that shows the importance of air quality in the IVF laboratory and its surrounding areas. In recent years, newly built ART centres are outfitted with HEPA and VOC filtration units with the laboratory and operating rooms over pressurized to exclude contamination from the surrounding areas. Existing units are also being renovated to retrofit their spaces or outfit their working areas with portable filtration systems. Among the many changes in the field of assisted reproductive technology, the implementation of air filtration units has certainly increased and improved the outcomes and utilization of the technology.


References

ASRM. Revised minimum standards for practices offering assisted reproductive technologies (2008) Fertil Steril 90, S1 65-68

Brown SK (1999) Chamber assessment of formaldehyde and VOC emissions from wood-based panels. Indoor Air 9, 209-215.

Cohen J, Gilligan A, Esposito W, Schimmel T and Dale B (1997) Ambient air and its potential effects on conception in vitro. Hum Reprod 12 No. 8, 1742-1749.

Cohen J, Gilligan A and Willadsen S (1998) Culture and quality control of embryos. Hum Reprod 13 Suppl 3, 137-44.

De Bortoli M, Kephalopoulos S, Kirchner S, Schauenburg H, and Vissers H (1999) State-of-the-art in the measurement of volatile organic compounds emitted from building products: results of European inter laboratory comparison. Indoor Air 9, 103-116.

Esteves SC and Bento FC (2013) Implementation of air quality control in reproductive laboratories in full compliance with the Brazilian Cells and Germinative Tissue Directive. Reprod Biomed Online 26, 9-21.

European Union 2004a Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage, and distribution of human tissues and cells. Official Journal of the European Union, L 102/48. European Union

Forman M, Polanski V, Horvath P, Gilligan A and Rieger D (2004) Reductions in volatile organic compounds, aldehydes, and particulate air contaminants in an IVF laboratory by centralized and stand-alone air filtration systems. Ferti. Steril. 82 Suppl 2, S324 (Abstract P-535).

Gilligan A, Schimmel T, Esposito Jr B, and Cohen J (1997) Release of volatile organic compounds such as styrene by sterile petri dishes and flasks used for in-vitro fertilization. Ferti. Steril. 68 Suppl 1, S52-53.

Gilligan A (2010) Establishing the IVF Laboratory: A Systems View. In ‘Reproductive Endocrinology and Infertility’. (Eds DT Carrell and CM Peterson) pp. 569-578. (Springer: New York)

Hall J, Gilligan A, Schimmel T, Cecchi M and Cohen J (1998) The origin, effects and control of air pollution in laboratories used for human embryo culture. Hum Reprod 13 Suppl 4, 146-55

Heitmann RJ, Hill MJ, James AN, Schimmel T, Segars JH, Csokmay JM, Cohen J and Payson MD (2015) Live births achieved via IVF are increased by improvements in air quality and laboratory environment. Reprod Biomed Online 31, 364-371.

Khoudja RY, Xu Y, Li T and Zhou C (2013) Better IVF outcomes following improvements in laboratory air quality. J Assist Reprod Genet 30, 69-76.

Johnson JE, Boone WR, and Bernard RS (1993) The effects of volatile compounds (VC) on the outcome of in vitro mouse

embryo culture. Fertil Steril Suppl 1, S98-S99, Abstract P-038.Lawrence, C et al. (2007) VOC Levels in a new IVF Laboratory with

both central and in-laboratory Photocatalytic air purification units. Alpha (Scientists in Reproductive Medicine) No. 36

Legro RS, Sauer MV, Mottla GL, Richter KS, Li X, Dodson WC and Liao D (2010) Effect of air quality on assisted human reproduction. Hum Reprod 25, 1317-24.

Llosá V, Lopez L, Rivero P and Palumbo A (2004) Fertilization, implantation, and pregnancy rates before and after the installation of Coda® incubator units in a human IVF laboratory. Unpublished.

Locke DC (1990) Determination of Cl through C5 Atmospheric Hydrocarbons. In Lodge Jr. J.P. (ed). Methods of Air Sampling and Analysis. Lewis Publishers, p.243.

Lundgren B, Jonsson B, and Ek-Olausson B (1999) Materials emission of chemicals - PVC flooring materials. Indoor Air 9, 202-208.

Mayer JF, Nehchiri F, Weedon VM et al. 1999 Prospective randomized crossover analysis of the impact of an IVF incubator air filtration system (Coda, GenX) on clinical pregnancy rates. Fertil Steril 1, S42–S43.

Merton JS, Vermeulen ZL, Otter T, Mullaart E, de Ruigh L, Hasler JF (2007) Carbon activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos. Theriogenology 67:1233–1238

Morbeck DE (2015) Air quality in the assisted reproduction laboratory: a mini-review. J Assist Reprod Genet 32, 1019-24.

Mortimer D (2005) A critical assessment of the impact of the European Union Tissues and Cells Directive (2004) on laboratory practices in assisted conception. Reprod Biomed Online 11, 162-76.

Munch E, Sparks A, Duran H and Van Voorhis B (2015) Lack of carbon air filtration impacts early embryo development. J Assist Reprod Genet 32, 1009-1017.

Racowsky C, Jackson KV, Nurredin A, Balint C, Shen S, de los Santos MJ, Kely JR and Pan Y (1999) Carbon-activated air filtration results in reduced spontaneous abortion rates following IVF. In ‘Proc. 11th World Congress on In-Vitro Fertilization and Human Reproductive Genetics’. Sydney, Australia. (Ed.) pp. O-059 (Abstract).

Schimmel T, Gilligan A, Garrisi GJ, Esposito Jr B, Cecchi M, and Dale B (1997) Removal of volatile organic compounds from incubators used for gamete and embryo culture. Ferti. Steril. 68 Suppl 1, S165.

Sparks LE, Guo Z, Chang JC, and Tichenor BA (1999) Volatile Organic Compound emissions from latex paint - Part 1 - Chamber experiment and source, model development. Indoor Air 9, 10-17.

Van Voorhis BJ, Thomas M, Surrey ES and Sparks A (2010) What do consistently high-performing in vitro fertilization programs in the U.S. do? Fertil Steril 94, 1346-9.



Don’t leave it to chancebe prepared with Coda®.

The Coda® Inline® Filter patented technology is proven to effectively remove VOCs and CACs. They are CE certified and manufactured in a clean room. Each Coda® Inline® Filter contains a HEPA filter and can last between 3-6 months. All Coda® Inline® Filters are FDA 510(k) cleared, ISO 13485:2003 medical certified and made in the USA.

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LifeGlobal® Family of Oils

THE QUALITYYOU CHOOSEMATTERS

LifeGlobal® Oils are intended for use as an oil overlay for droplets of medium for embryo culture or manipulation.

• High Quality, Washed and Sterile

• Pharmaceutical Grade Oils

• Extensively Tested

• Clean Oil with Clean Records

• 1-cell MEA and Endotoxin Tested

• Optimal Viscosity for Embryo Culture

• 24 Month Shelf Life

• Specifically designed & tested for IVF

• FDA Cleared & CE Marked

PURE OIL, PURE CONSISTENCY


µDrop GPS® DishEmbryo Culture Dish for Human IVF

Safe Effective Easy-to-use

New Design Features• Precise 20 µl micro-wells with GPS® feature for rapid location

and visualization• Enhanced optics• Better orientation and identification• EmbryoAddressTM helps to quickly identify and track embryos • 32 mm inner ‘oil ring’ for VOC protection • Uses up to 75% less oil than a conventional 60 mm dish• Fill the outer wells with oil for better temperature stability out of

the incubator• Works well with both oil-overlay and oil-underlay methods• Designed to ensure safe embryo culturing• For use with both standard and mini incubators

μDrop GPS®

General Features of GPS® Dishware• Improved safety (no droplet collapsing or mixing)• Better pH control• GPS® microwells protect against sudden movements• Raised lid promotes gas exchange, prevents contamination,

and stops oil seals from forming• Non-toxic medical grade, non-pyrogenic olystyrene• Better ergonomics than a 35 mm dish• CE and ISO Registered, FDA 510(k) Cleared• 1-cell MEA and LAL Tested

New Breathable Packaging for all GPS® Dishware

• Reduces off-gassing time• Less VOCs introduced into the laboratory • Tested and validated to maintain sterility for the entire 5-year

shelf life of the dish

“The Micro GPS dish evolved from the input of dozens of scientists and engineers. It is the most versatile and ingenious culture dish ever developed. It combines so many new features.

It is the strongest and safest Petri dish for culturing embryos. The bottom underneath each GPS location is so thin, you can see the embryo smile.”

New Design FeaturesThe working surfaces (bottom) of all wells of the μDrop GPS® dish is sloped to create the GPS® location for all the specimens within these wells. This makes locating them quicker, easier to handle and observe, and the ability to return specimens to their controlled environment sooner.= Egg or Embryo

The working surface and well bottoms

Jacques Cohen, PhD

LifeGlobal® Family of Oils

THE QUALITYYOU CHOOSEMATTERS

LifeGlobal® Oils are intended for use as an oil overlay for droplets of medium for embryo culture or manipulation.

• High Quality, Washed and Sterile

• Pharmaceutical Grade Oils

• Extensively Tested

• Clean Oil with Clean Records

• 1-cell MEA and Endotoxin Tested

• Optimal Viscosity for Embryo Culture

• 24 Month Shelf Life

• Specifically designed & tested for IVF

• FDA Cleared & CE Marked

PURE OIL, PURE CONSISTENCY


global®The leading scientifically based and clinically proven

‘Uninterrupted Single Solution Culture Medium®’

Let the Embryos Choose! ®

Based on Pure Science

v The First leading scientifically based ‘Single Solution Medium®’

v Proven to work with any type of VOC controlled environment as a continuous culture

v Over 150 Independent Publications using global® medium

v Over 15 years of Consistent Superior Results worldwide

‘Uninterrupted Time-Lapse Culture Medium®’


“We are extremely happy with Global medium combined with the Embryoscope. Nice blastocyst formation and our pregnancy rates have been over 70% with Day 5 transfer. We use it as a continuous culture medium from Day 1 onwards with no media exchange.”

“We transitioned from using it from D1 thru 6 with a half change on Day 3 to using it for “uninterrupted culture” from Day 1-Day 6. Equivalent results both ways.”

Nina Desai, PhD, HCLD, Cleveland Clinic

(global® user since 2001. No commercial ties with the company.)

1. Uninterrupted culture of human embryos in global® or global® total®

• CampbellA,FishelS,BowmanN,DuffyS,SedlerMandHickmanCFL(2013)Modellingariskclassificationofaneuploidyinhuman embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85.

• Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with donor oocytes. Human Reprod. 28 Suppl. 1, i184 (Abstract P-167).

• Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Supplement, S6 (Abstract O-5).

• Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381.

• Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6).

• Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800.

• Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during controlledovarianstimulationinfluencesearlyembryodevelopmentalkinetics:atime-lapsestudy.Eur J Obstet Gynecol Reprod Biol 168, 167-72.

2. Time-lapse culture of human embryos in global® or global® total®

• Basile N, Morbeck D, Garcia-Velasco J, Bronet F and Meseguer M (2013) Type of culture media does not affect embryo kinetics: a time-lapse analysis of sibling oocytes. Hum Reprod 28, 634-641.

• Bellver J, Mifsud A, Grau N, Privitera L and Meseguer M (2013) Similar morphokinetic patterns in embryos derived from obese and normoweight infertile women: a time-lapse study. Hum Reprod 28, 794-800.

• CampbellA,FishelS,BowmanN,DuffyS,SedlerMandHickmanCFL(2013)Modellingariskclassificationofaneuploidyinhuman embryos using non-invasive morphokinetics. Reprod Biomed Online 26, 477-85.

• Campbell A, Fishel S, Bowman N, Duffy S, Sedler M and Thornton S (2013) Retrospective analysis of outcomes after IVF using an aneuploidy risk model derived from time-lapse imaging without PGS. Reprod Biomed Online 27, 140-6.

• Costa-Borges N, Bellés M, Herreros J, Teruel J, Ballesteros A, Pellicer A and Calderón G (2013) Single medium culture in a time-lapse incubator until the blastocyst stage with or without medium renewal on Day-3: a prospective randomised study with donor oocytes. Human Reprod. 28 Suppl. 1, i185 (Abstract P-167).

• Cruz M, Garrido N, Herrero J, Perez-Cano I, Munoz M and Meseguer M (2012) Timing of cell division in human cleavage-stage embryos is linked with blastocyst formation and quality. Reprod Biomed Online 25, 371-381.

• Desai NN, Ploskonka S, Goldberg J, Austin C and Falcone T (2013) Morphokinetic analysis of embryos from patients having a day 5 transfer: preliminary results with the embryoscope. Fertil Steril 100, S120 (Abstract O-392).

• Martinez-Burgos M, Losada C, Pareja S, Agudo D and Bronet F (2013) Effects of low O2 concentration in extended embryo culture using benchtop incubators (Embryoscope and MINC). Fertil Steril 100, S251 (Abstract P-360).

• Munoz M, Cruz M, Humaidan P, Garrido N, Perez-Cano I and Meseguer M (2013) The type of GnRH analogue used during controlledovarianstimulationinfluencesearlyembryodevelopmentalkinetics:atime-lapsestudy.EurJObstetGynecolReprodBiol 168, 167-72.

• Nakahara T, Iwase A, Goto M, Harata T, Suzuki M, Ienaga M, Kobayashi H, Takikawa S, Manabe S, Kikkawa F and Ando H (2010) Evaluation of the safety of time-lapse observations for human embryos. J Assist Reprod Genet 27, 93-6.

• Ramirez JM, Fernandez FG, Bueno AS, Brandt M, Fernandez JAG and Lopez EG (2012) Importance of multinucleation at 2-cell stage: study in a time-lapse incubator. Fertil Steril 98 Suppl., S-169 (Abstract P-196).

• Semeniuk L, Mazur P, Mikitenko D, Nagorny V and Zukin V (2013) Time-lapse and aCGH, is there any connection between ploidy and embryo cleavage timing on early stages of embryo development? Fertil Steril 99 Suppl, S6 (Abstract O-5).

• Silva MM, Llanos BA, David Gumbao, Marcos J, Sanchez A, Nicolas M, Olmedilla LF and Gutierrez JL (2012) Optimization of clinical outcomes in an oocyte donation programme. Reprod Biomed Online 24 Suppl 1., S7 (Abstract PP-6).


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Since its introduction in 1989, pre-implantation genetic diagnosis (PGD) has been used to evaluate the genomic characteristics of human embryos and to prevent

the transmission of inheritable diseases, representing an alternative to prenatal testing. In general, PGD comprises primer-based DNA amplification for single gene disorders or structural chromosome alterations. A similar approach to PGD, pre-implantation genetic screening (PGS) has also been established in IVF as a valuable tool for the identification of aneuploid embryos, which are known to have little potential to result in a viable pregnancy, but cannot be distinguished from normal embryos using standard morphological evaluation. Various studies and robust data suggest that the use of PGS may significantly improve embryo implantation rates, particularly, in patients of advanced maternal age or those who have experienced recurrent miscarriages or implantation failures in previous cycles.

In the past, the genetic analysis of oocyte polar bodies was primarily used for aneuploidy screening, while embryo biopsy at cleavage stage, which involves the removal of one or two blastomeres from pre-compaction Day 3 embryos, has been a preferred practice for PGD and PGS. The analysis of the single cells (polar bodies or blastomeres) for numeric or structural chromosomal abnormities used to be performed by fluorescence in-situ hybridization (FISH). However, the potential benefit of FISH for genetic screening has been questioned in a number of prospective randomized studies that provided disappointed clinical results. Its reduced diagnostic value has been associated with the fact that it only allows the analysis of a limited number of chromosomes and it is performed in a single cell which may not be representative of the entire embryo.

New technologies for 24-chromosomes analysis have then been proposed and are currently available

for clinical use, including array comparative genomic hybridization (array-CGH), metaphase comparative genomic hybridization, single-nucleotide polymorphism micro-arrays, quantitative polymerase chain reaction and next-generation sequencing (NGS). Although very

powerful, the limited amount of material (one or two cells) obtained for genetic analysis resulting from polar body or cleavage stage embryo biopsy techniques can also limit the accuracy of these techniques. Therefore, to overcome these technical limitations and eventually increase the sensitivity and reliability of the genetic diagnosis, another biopsy technique based on the excision of trophectoderm cells, ideally between 4 and 8, from a blastocyst has been proposed. This technique represents some benefits compared to polar body or blastomere biopsy, as it allows the analysis of more cells and

increases the possibility to detect the presence of mosaicism in the analyzed embryo.

At present, IVF centres have been gradually introducing blastocyst-stage biopsy into their clinical practice, even though it may be technically challenging. It is noteworthy that trophectoderm biopsy is usually performed on Day 5 or Day 6 of development, and the time needed to obtain the results from the genetic analysis may not allow a fresh embryo transfer. Additionally, it is essential that IVF labs have a well-established, stable extended embryo culture system and a good cryopreservation program to guarantee that blastocysts can be obtained, biopsied, vitrified, and, subsequently, thawed and transferred successfully in a subsequent cycle.

The technical difficulties experienced during trophectoderm cell excision may vary from blastocyst to

Trophectoderm Biopsy: The Pulling and Flicking Techniquesby Nuno Costa-Borges*, PhD and Gloria Calderón, PhD*Scientific Director, Embryotools SL, Barcelona, Spain

You can contact Nuno Costa-Borges at [email protected]

nuno coSTa-BorGeS

Since its first introduction in 1989, pre-implantation genetic diagnosis (PGD) has been used to evaluate the genomic characteristics of human embryos and to prevent the transmission of inheritable diseases, representing an alternative to prenatal testing.


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blastocyst depending on their quality, grade of expansion, and position of the inner cell mass (ICM), among other details. It is therefore recommended that embryologists acquire extensive training in this technique using animal models, such as mouse or cattle blastocysts before implementing the technique into the routine of the human IVF lab.

In our practice, we use the so-called “pulling” and “flicking” methods, which allow us to perform trophectoderm biopsies on virtually all types of hatching or fully hatched blastocysts. Although technically demanding, these two techniques have small details and tricks that can greatly simplify and reduce the expected technical challenges.

The “pulling” method is particularly indicated for hatching blastocysts with only a few TE cells herniating through the zona pellucida. As a first step, blastocysts are examined under the inverted microscope and rotated until the ICM is clearly identified. The blastocyst is then oriented so that the herniating TE cells face the biopsy pipette. At this point, the blastocyst is held firmly with the holding pipette and the biopsy needle is brought near the TE cells, which can then be drawn carefully into the biopsy pipette by applying gentle suction. Subsequently, the TE cells are pulled away from the blastocyst, while laser pulses are applied. The detachment of the TE cells should normally be achieved with less than five laser pulses. It is important to be careful with the amount of pressure exerted inside the biopsy capillary so not to lose control of the TE cells post-biopsy. For a successful excision of the cells, laser pulses of the optimum intensity should be applied at the strategic spots of the embryo, so they can easily be pulled away from the blastocyst while minimizing cell damage. An example of a trophectoderm biopsy performed using the “pulling”

Figure 1. Trophectoderm biopsy using the “pulling” technique, indicated for an early hatching blastocyst.

method is illustrated in Figure 1.The “flicking” method is recommended for blastocysts

with TE cells herniating through a wide zona pellucida opening or for completely-hatched blastocysts. As described above, all blastocysts must be assessed morphologically under an inverted microscope and rotated if necessary until the ICM is clearly identified. The blastocyst should be positioned so that the ICM is next to the holding pipette (Figure 2). TE cells located on the opposite side of the ICM can then be carefully aspirated into the biopsy pipette. Immediately after, a first laser pulse is applied to one edge of the trophectoderm, followed by two or three more laser pulses. While laser pulses are applied, the blastocyst should collapse, allowing more TE cells to be drawn inside the biopsy pipette. The collapsed blastocyst is then gently released from the holding pipette while the biopsy pipette holds the embryo through the TE cells. Immediately, the holding and biopsy pipettes should be aligned on the same focal plane and TE cells can then be detached from the blastocyst with a quick flicking movement of the biopsy pipette against the holding pipette (Figure 2).

Once biopsied, the blastocysts can be vitrified while still collapsed or put back in optimal culture conditions. The biopsied cells should be transferred from the micromanipulation dish into an Eppendorf tube. This part of the process, generally known as “tubing of cells”, should be performed in a DNA-free environment, under a stereomicroscope. To ensure that the biopsied cells are correctly deposited inside the Eppendorf tube, the cells must be viewed to confirm that they come out of the capillary. Commonly, embryologists face problems in focusing on the tip of the capillary at the same time as the cells are transferred into the bottom of the Eppendorf tube. To solve this problem we recommend using an adaptor (e-tubingTM)


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Figure 2. Biopsyprocedureusingthe“flicking”method.

Figure 3. The “tubing” process of transferring the biopsied cells into an Eppendorf tube under a stereomicroscope.

that has been developed to hold the Eppendorf tubes at different angles and simplify the technique. This has proved to be of great assistance to embryologists (Figure 3).

In conclusion, with the appropriate equipment and the correct technique for blastocyst culture and cryopreservation, blastocyst biopsy can represent a practical and preferable path to pre-implantation genetic testing of

embryos, compared to polar body or cleavage stage embryo biopsy. Trophectoderm biopsy is likely to become the gold standard to obtain material for pre-implantation genetic testing. However, fully mastering this technique requires appropriate training and micromanipulation skills and choosing the right tools is an essential pre-requisite for obtaining consistent and successful results.


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Effect of Coda® Air Filtration onChemical and Clinical Pregnancy Rates

(Battaglia et al., Fertil Steril 75, Suppl. 1, 6S, 2001)

Effect of Follicular Phase Particulate AirPollution on Pregnancy Loss

(Perin et al., Fertil. Steril., 2009, in press)

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transfer of in vitro-produced bovine embryos

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ReproductiveBioMedicine

Online

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An international journal devotedto biomedical research on human conception and the welfare of the human embryo

VISIT OUR RESOURCE CENTERS

on www.rbmonline.com IFFS-UIT Resource Center

Serono Symposia International Foundation Resource CenterBob Edwards Resource Center

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American College of Embryology(ACE)

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International Society forFallopian Tubes and

Reproductive Surgery (ISFT-RS)

International Society for In Vitro Fertilization (ISIVF)

Mediterranean Society for Reproductive Medicine (MSRM)

Preimplantation GeneticDiagnosis International Society

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Turkish Society of Reproductive Medicine (TSRM)

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ReproductiveBioMedicine

Online

www.rbmonline.com

An international journal devotedto biomedical research on human conception and the welfare of the human embryo

VISIT OUR RESOURCE CENTERS

on www.rbmonline.com IFFS-UIT Resource Center

Serono Symposia International Foundation Resource CenterBob Edwards Resource Center

Alpha (Scientists inReproductive Medicine)

American College of Embryology(ACE)

The Global Chinese Associationfor Reproductive Medicine

(GCARM)

International Society forFallopian Tubes and

Reproductive Surgery (ISFT-RS)

International Society for In Vitro Fertilization (ISIVF)

Mediterranean Society for Reproductive Medicine (MSRM)

Preimplantation GeneticDiagnosis International Society

(PGDIS)

Turkish Society of Reproductive Medicine (TSRM)

Affiliated Societies


ARTICLES

Introduction

Successful pregnancy requires the coordination of an array of signals and molecules from multiple tissues that can be compromised by several stress agents. Reproductive health problems and subsequent treatment can be a source of distress such that 20-25% of the women experience minor to severe distress, which can either reduce quality of life or efficacy of continuation of reproductive techniques (Verhaak et al., 2010; Gameiro et al., 2013). Natural interventions to reduce stress have been shown to have positive effects on pregnancy rates, without harm or unintended effects on assisted reproduction (Kiltz, 2014; Van Dogean et al., 2016). Non-invasive techniques (e.g. IUI) will provide a lower level of distress when compared to invasive techniques. IVF/ICSI success rates are much higher than they were before compared to IUI that has not been changed. Although IUI is widely practiced, the technique is still used as an empiric treatment with little evidence of effectiveness (5 to 17%), directly influenced by the laboratory standards (Rao 2014).

Stress connection

It has already been demonstrated that natural therapies such as psychological support and exercize reduce stress and improve empowerment in patients undergoing surgery or chemotherapy (Braun, 2016; Van den Berg et al., 2012). The link between infertility and stress is also well recognized, confirming that under a lower level of stress the conception rates are higher (Domar et al., 2000).

Emotional distress produces toxic markers that, at a high level, may negatively interfere with the communication between the endometrium and the embryo, increasing the chance of implantation failure and miscarriage. Detecting and addressing emotional distress is required for patients and recommended by the ESHRE 2015 guideline. To reduce stress during fertility care is paramount to create a relationship of trust among doctor, patient and paramedical group; whereas, full anamneses and precise diagnostic will support the patient’s expectation. In addition, the study of the reproductive system under lower-level distress is important to better understand the ideal scenario for a successful pregnancy.

Integrated care

Physicians, psychologists, embryologists, nurses, laboratory technicians, natural therapists and others actively support the field of reproductive medicine and should be part of the care team. The group should have a comprehensive understanding of the patient’s prognosis in order to ensure the right fertility treatment incorporated with an alternative plan with one goal: improve daily life’s behavior to enhance conception. Alternative treatments address a variety of conditions associated with reproduction including stress and anxiety reduction that is fundamental to, regulating the menstrual cycle with better accuracy of ovulation timing in natural and modified-natural stimulation, improving egg and sperm quality during the three months prior to conception, improving blood flow in the uterus, decreasing the chance of miscarriage and others. It is important to meet the patients’ needs; consequently, more therapy choices are offered with better personalized plans. These integrative therapies may include, lifestyle coaching, exercises, nutritional counseling, acupuncture, massage, art therapies, and others that could be offered in isolated or combined therapies.

Increasing effectiveness and decreasing costs

The prevention of infertility is still the most important and cost-effective treatment strategy that could be supported by the members of integrative care. Low-cost ovarian stimulation protocols encourage the use of natural cycles, modified-natural cycles and mild ovarian stimulation. Improving IUI efficiency is a requisite to avoid more costly and invasive procedures that will contribute to increasing patient compliance and avoid discontinuation of IVF treatment. Everyone will benefit and the budget, normally offered by the national healthcare, will be better allocated among the target population. It is essential to work up with eligible couples (e.g. woman less than 40 years with good ovarian reserve, normal or mild male factors infertility) in order to reach higher effectiveness using IUI. Because of the differences among patients, standard management should be avoided. A personal program is required either to

Optimizing success in reproductive techniques: decreasing emotional distress by improving personal care.by Ana Rita S. Coutinho, clinical embryologist certified (CFAS), MDV, PhDFounder and Scientific Director for Animus Biotechnology,Montreal, Quebec, Canada.

You can contact Ana Rita S. Coutinho at [email protected]

ana riTa S. couTinho, MDV, PhD


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monitor the follicular phase or to optimize sperm washing to reach the ideal amount of motile sperm (TCM between 10 to 20 million/mL).

Conclusion

Personalized care in assisted fertility is recommended and promising. Improving patients’ supervision will create a trusted environment with the active partnership among the mains actors (physician-patient-paramedical) that will reduce distress levels in treated patients with a positive effect on assisted reproduction. In parallel, non-invasive treatment will offer a natural model to investigate the reproductive pathways using a model closer to the physiological system using minimal hormonal stimulation. Although developments in ART have led to numerous interventions designed to improve human fertility, infertility care is not a priority in almost all reproductive health care centres. Consequently, further investigation to improve fertility programs’ success and feasibility must be achieved in multiple places and countries in order to improve participation and generalization.

References

Domar, A.; Clapp, D.; Slawaby, E.; Dusek, J.; Kessel, B., Freizinger, M (2000) Impact of group psychological interventions on pregnancy rates in infertile women. Fertility and Sterility 73(4): 805-811.

Braun, D (2016) Cancer therapy. In. ‘The New Yorkes’, pp.9-10.Gameiro, S.; Boivin, J.; Domar, A (2013) Optimal in vitro

fertilization in 2020 should reduce treatment burden and enhance care delivery for patients and staff. Fertility and Sterility 100: 302-308.

Kiltz, R. (2014) The fertile secret: an integrative approach to fertility care. In. ‘Fertility Magazine’. pp. 36-37.

Rao, K.; Carp, H.; Fisher, R (2014) Principles & practice of assisted reproductive technology, 632-649 (Jaypee brothers medical publishers)

Van Dongen, A.; Nelen, W.; IntHout, J.; Kremer, J.; Verhaak, C (2016) e-Therapy to reduce emotional distress in womwn undergoing assisted reproductive technology (ART): a feasibility randomized controlled trial. Human Reproduction 1: 1-12.

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IVF Data Flow: From Trash Bins to Clipboards to EMR to Smart Appsby Stephen Fiser, Giles Tomkin and Jacques Cohen

You can contact Jacques Cohen, PhD at [email protected]

JacqueS cohen, PhDA large amount of paperwork is produced in clinical

medicine (Woolhandler and Himmelstein, 2014). When only paper is used, the task of organizing

and controlling patient treatment records is onerous and subject to varying degrees of error. Physicians can spend more than 20% of their time shuffling paper charts and reports. Scrutiny and oversight through non-governmental and governmental inspections and audits have clearly increased this burden. This is especially true in assisted reproduction where laboratory staff may spend even more time on paperwork than other clinical team members. It is estimated that only 30% of IVF clinics have implemented an electronic medical record (EMR) system, although this figure is difficult to confirm.

Assisted reproduction is the only medical intervention that is designed to treat not only individuals, but couples, or even multiple participants, perhaps including gamete donors and gestational carriers. To make matters even more complicated, these multiple individuals have associated with them gametes and embryos, whose development must be monitored and recorded meticulously during several days in the laboratory. All this monitoring generates staggering amounts of information and paperwork for each patient and procedure. Yet, order can be established in what may look to the unfamiliar like an impossible system integrating patients, gametes, embryos, lab procedures, assays, surgery, counseling and more. Patients often undergo multiple attempts, but what may be considered standard treatment in one clinic or country might be illegal or considered unconventional, in another locale. Generally speaking, there are three streams of paperwork, more or less integrated.

The Electronic Medical Record (EMR)

The first data stream involves patient procedures and attempts to achieve pregnancy. This includes monitoring follicular development in the female partner, administration of drugs, and surgical steps. Later, individual gametes and embryos are tracked, although, in many labs, embryos are

grown in groups so that individual identity within the cohort is lost. This flow of information can be captured with a tailored EMR system, also referred to as an Electronic Health record. An EMR is an ordered collection of patient health information data in a digital format. It becomes increasingly complex when records are shared across different health care settings. Sometimes this can take the form of an entire country’s health care records (for example, national registries in Sweden, France, Belgium, The Netherlands and other countries). Records are shared through network-connected information systems. The EMR

may include a wide range of patient information, including personal data and demographics, medical history, medication and allergies, vaccination status, laboratory test results, radiology images, vital signs, and billing information. EMRs for assisted reproduction have the added complexity of tracking two or more individuals throughout their treatment and following their eggs, sperm and resulting embryos

individually or in groups. The beginning of treatment is quite clear and data entry is usually straightforward, but because gametes and embryos can be cryopreserved and later thawed across multiple attempts and for different purposes, the end of treatment is often unclear and final embryo dispositions can be postponed. Existing EMR systems generally do not have automatic error detection capabilities and cannot easily flag incorrect or missing data; some systems also lack detailed reporting features. These deficiencies are especially perplexing because subscription fees to EMR systems are quite high, which inevitably lead to increasing costs to the patient. Moreover, laboratory instrumentation tracking and possible correlation with outcomes is lacking in most EMR’s. ART, oddly enough, has not yet embraced improvements in data handling through

Physicians can spend more than 20% of their time shuffling paper charts and reports.


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Applications (Apps), but more on that later.

EMR systems should be designed to collect and store all relevant data and to record the status of a patient’s attempts at pregnancy across time. They should obviate the need to track down and search a patient’s paper records for information, particularly if the electronic data are systematically verified and known to be accurate. EMRs should reduce data duplication, keep the patient file up to date, and decrease the risk of information loss. A major advantage of EMRs is that digital data are searchable, analyzable and can be copied at any time. This is achieved by reporting and filtering features, which should always be integral to the EMR. Unfortunately, this ideal scenario is often not the case, and users may complain about either the lack of reporting features or their impracticality. Due to digital sorting and searching, EMRs are very effective when extracting medical data for the examination of possible trends and fluctuations in outcomes over time. An EMR without search features and sophisticated reporting is narrow and not much more useful than a written record system. EMR user satisfaction levels are mostly anecdotal since no systematic surveys have been done. Many ART EMRs are based on outdated computer languages, data input, and sorting systems. Flexibility and the ability to redesign and change types of data entry in the future should be key when designing EMRs for ART.

Enterprise Software

The second stream of IVF information concerns organizational and administrative data, such as that of any large organization or enterprise. Enterprise software satisfies the needs of the organization rather than those of individual users. Such organizations include businesses, manufacturing plants, schools, charities, governments, universities and clinics. Services provided by enterprise software are typically business-oriented tools such as online purchasing and payment processing, automated billing, security, enterprise and IT management, client services, project, collaboration and human resource management, manufacturing, occupational health and safety, application integration, and forms automation. Many of these aspects are needed in the IVF environment, however rarely are any such enterprise systems applied to independent IVF clinics, due to high cost and the need to train and test staff. Enterprise software is often available as a suite of customizable programs. The complexity of these tools and the entire system requires expertise and specialized knowledge, often being very expensive.

Laboratory and Program Quality Management

The third information stream in ART is the quality management (QM) program for andrology, embryology,

and chemistry laboratories. Electronic record keeping is mostly lacking in this area. Quality control (QC) of laboratory instruments and activities is most commonly achieved through multiple home-made forms, filled in by hand on a daily basis, or, less often, entered directly into an Excel sheet, and very rarely compiled in a way that allows effective and timely reporting. QC data review typically occurs on a monthly basis and the paperwork is only partially tracked and audited during annual laboratory inspections. Rarely do labs scrutinize the data to gain insight into instrument performance unless some catastrophic event occurs. This is partly because the format of data collection makes such an effort unreasonably time-consuming, mostly uninformative, and difficult. Fluctuations in instrument performance may therefore be overlooked. Filing handwritten forms in cabinets or binders is an outdated system, which basically means that large amounts of important data are being discarded.

New Opportunities

Worldwide, 2.5 quintillion bytes of data are created every day. That means that 90% of the world’s data has been created within the past two years (source IBM). This has placed an incredible demand on technologies capable of storing and analyzing these data. These new technologies, in combination with the explosion of mobile devices, have created an enormous opportunity to improve the state of affairs in the laboratory. Instead of writing everything down on forms attached to a clipboard and entering the data into an outdated system at a later point in time (or usually not), laboratory staff should be able to enter or dictate the data on a phone or tablet while they work – mostly eliminating errors that occur due to a messy, multistep process – and generate a searchable, permanent, and immediately useful electronic record.

Rightly or wrongly, there are no international and uniform requirements for the setup and maintenance of laboratories and equipment. This means that any existing software will be suitable for some situations but not for others; which in turn means the data generated by these varied programs will be too heterogeneous for comparison. Each clinic will follow its own standards.

Our new startup called IVF-QC recently released an App (Reflections™ by IVFqc) that allows customization of quality control for any laboratory instrument and associated parameters (http://ivfqc.com). The App is accessible from any internet-connected device (computer, tablet or smartphone of any make) and not only does away with the clipboard, but also allows for real-time fluctuation tracking and detailed reporting. The App can be applied to any QC program in any type of laboratory and is malleable to almost any laboratory system. It has a so-called generic


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JSON API, which means that it allows connection to other software and hardware with relative ease. Some early adopters of the Reflections™ App are laboratory directors who run multiple laboratories and can now conveniently and efficiently track each of their laboratory’s QC activities from their phone, jumping from lab to lab with just a few clicks. The standard of security is based on HIPAA, as well as two-factor authentication and double encryption. The App’s second version is slated for release by the end of 2016 and will have an “action package” such as the ability to send useful notifications and reminders to users within a team. It will also allow for tracking instrument performance within a given acceptable range. In this way, a subtle drift upwards, for instance, of temperature within the established range of temperature in an incubator can be spotted before it becomes problematic. Such shifts can provide early warning of failure for many kinds of instruments.

Many different types of laboratories can benefit from using Reflections™ (https://www.reflectionsapp.net). The application has a sign-up and a sign-in section, including secure dual log-in, similar to banks. Once logged-in, users can add one or more new labs, and add members with specific assigned access levels. When a new lab is added, new instrument types (or groups) are added, followed by individual instruments within each group. For example, in the embryology laboratory, instrument groups could include large box incubators, bench-top incubators, warmers, hoods, Dewars, refrigerators, etc. and each group can include individual makes and models with different QC requirements. Within each instrument, parameters can be created and their acceptable normal ranges added. For example, for incubators, CO2 and O2 levels, temperature, and humidity, display and measured, may be added with specific acceptable ranges for each of these parameters, which can be changed later if needed. When the instrument groups and instruments are in place, users can create one or more data logging plans, which can be set for desired intervals including daily, weekly, monthly, etc. These too can be edited later as desired. Data entry can be performed using any tablet, notebook or regular computer with internet access, though hand-held devices are preferable for their portability. Data review can take place at any time from anywhere, which is particularly useful for organizations with multiple laboratories dispersed in different rooms, buildings, cities or even countries. Directors and supervisors

can check for data completeness and review data via their browser on any internet-connected device including their cell phones. Summary reports can be generated and viewed by staff members with permitted access. These can be downloaded as PDF for printed records and emailed to overseers as required. Alternatively, the reports may be electronically signed from any location. Fluctuation and detail reporting shows all entries made for any instrument within any instrument type, so that any instrument’s recorded data can be seen for any time period as a graph, giving staff the opportunity to see early indications of instrument or system malfunction. Incident reporting allows for staff to enter important occurrences that merit further attention, reasonably divided into varying levels. This allows direct and effective follow-up according to urgency.

As can be seen from this brief description of the setup and use, the application Reflections™ has obvious advantages over, dare we say it, the clipboard or even traditional Excel data recording for all the reasons given above.

As far as we are aware, as of spring 2016, there are no other applications with such features. It is also the case that none of the existing Apps offer the total flexibility that is allowed by Reflections™. None offer the useful reporting functions that allow accurate real-time instrument monitoring. There are applications suitable for certain business entities that have some similar features, but they have limited flexibility and often are enormously expensive to purchase and maintain. Reflections™ can be used by a range of business entities that require quality control and management, from ART laboratories to diagnostic laboratories, even zoos and wineries, for about $1 per day. Entry-level use is free. Reflections™ provides a serious step towards the important but elusive goal of paperless quality management, while improving service to patients and lowering cost.

References

Woolhandler S, Himmelstein DU. Administrative work consumes one-sixth of U.S. physicians’ working hours and lowers their career satisfaction. Int J Health Serv. 2014;44(4):635-42

IBM. Bringing big data to the enterprise. http://www-01.ibm.com/software/data/bigdata/what-is-big-data.html

The Consistency of global® MediumDon Rieger, PhD

May 15, 2013

The following graphs show the results of the quality control measurements of the same 24 sequential lots of global® medium manufactured between May, 2012 and April, 2013

Third-party measurements of the pH of 24 sequential lots of global®. With 10 mg/ml protein, at 37°C and 5.5% CO2

The osmolarity of 24 sequential lots of global® measured by freezing-point depression

Third-party measurements of the endotoxin content of 24 sequential lots of global®

The results of third-party 1-cell mouse embryo assays of 24 sequential lots of global®


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Abstract

Objective: to evaluate the potential of the slower growing blastocyst to establish a clinical pregnancy if transferred on Day 5 prior to blastocyst formation or if the embryo is vitrified on Day 6 after blastocyst development.

Design: retrospective data analysis January 2015 to November 2015

Outcome measure: clinical pregnancy as defined by fetal heartbeat

Conclusion: embryos which fail to form blastocysts, at the time of embryo transfer on Day 5, or embryos which require culture until Day 6 for blastocyst vitrification should not be excluded from consideration due to the acceptable pregnancy rates that can be established in fresh transfer and equivalent pregnancy rates in vitrification.

Introduction

Extended embryo culture to the blastocyst stage allows for the selection of those embryos that can continue to develop after the embryonic genome activation that generally occurs on Day 3 of embryo development in humans. Approximately 50 -70% of the cleavage stage embryos will develop to a blastocyst by Day 5 or Day 6. This extended development in culture allows for improved embryo selection and allows for better selection for those patients wishing to transfer fewer embryos and thus avoiding a multiple pregnancy.1,2

Improvements in culture media, incubators, group culturing and time-lapse have permitted embryo culture to continue successfully to Day 5 for selection and embryo transfer.3 One study assessed if time-lapse could predict blastocyst formation based upon morphokinetic parameters up to Day 3 when poor spermatozoa were injected. Due to the influence of the paternal factors after Day 3, this study was unable to establish a prediction tool when male factor is present.4 Many other studies have been conducted to investigate the development of the cleavage stage embryo and what parameters could indicate which embryos would be most likely to form blastocysts. A prediction model would permit selection of embryos and embryo transfer

on Day 3 and avoid the extra consumables and time spent on extended culture to blastocyst. However, one study reported that only 51% of the embryos that were transferred on Day 5 would have been preselected on Day 3.1 Therefore it seems that the only reliable method for the best selection of embryos developing to blastocyst is to grow the embryo to Day 5 for transfer to the patient.

Occasionally the embryos from certain patients develop more slowly than those of other patients. It has been shown that chromosomal abnormalities will not necessarily impair embryo development and therefore these slower growing embryos cannot be classified as abnormal and should not be excluded from consideration.5 Some factors such as spermatozoa DNA fragmentation, culture media volume, culture media formulations and gamete diversity have all been shown to impact the rate of blastocyst formation.6,7,8

The dilemma that arises is what to do when faced with the slower development of blastocysts for a patient when having already committed to extended embryo culture. Do these slower growing embryos implant after Day 5 transfer and are they suitable for vitrification on Day 6 once the blastocyst stage has been achieved?

The objective of this study was to assess the performance of slower developing embryos in a clinical situation both in fresh embryo transfer and after warming and transfer.

Materials and Methods

Data were collected for all patients under 40 years of age treated between January 2015 and the end of October 2015 with a double embryo transfer in fresh cycles and for single and double embryo transfer in warmed cycles. The data were retrospectively compared and assessed by Chi-squared analysis.

Patient preparation

Fresh cycles: patients were stimulated with exogenous gonadotropins using a long agonist protocol or GnRH

The Slower Growing Embryo – Is it Suitable for Day 5 Embryo Transfer and Day 6 Vitrification?by Carole Lawrence, Laboratory Director, Pacific Centre for Reproductive Medicine, Burnaby, British Columbia, Canada

You can contact Carol Lawrence at [email protected]

carole lawrence


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antagonist protocol. Luteal phase support was either Crinone® 8% PV QD or Endometrin® 100 mg PV TID from Day 1 of embryo culture and continued at least until the date of the pregnancy test 14 days later. Clinical pregnancy was assessed as the appearance of a gestational sac on ultrasound and evidence of fetal cardiac activity.

Warmed cycles: patients were prepared using an Estrace® only protocol with Endometrin® or a Lupron-estrace protocol with Crinone®. Embryo transfer of the warmed embryos was performed on the sixth day of progesterone. Luteal phase support was continued until at least 9 days after embryo transfer when the pregnancy test was performed (serum hCG). Patients with a positive test were instructed to continue on luteal support until at least after the first viability ultrasound. Clinical pregnancy was assessed as the appearance of a gestational sac on ultrasound and evidence of fetal cardiac activity.

Embryo culture

After oocyte collection, 36 hours post 10,000 IU hCG, the oocytes were inseminated at 3-5 hours post collection by either conventional IVF or ICSI. Oocytes having two pronuclei at 16-20 hours post insemination were group cultured until the day of transfer, in COOK® Sydney IVF Culture media. Embryo observations were made each day and the embryo stage and quality were documented. Cleavage stage embryos were grading using a generalized scoring system from 1-5 with 1 being the best quality embryos. Blastocysts were scored using the system developed by Gardner and Schoolcraft. A medium change was performed on D3 of culture and on Day 5 of culture. Assessment of embryos for transfer on Day 5 was performed one hour prior to the transfer procedure. The most advanced and best morphological quality embryos were selected for embryo transfer.

Vitrification

Excess embryos were cultured until the time of blastocyst formation, either later on Day 5 or Day 6, and vitrified using the Vitrolife Rapid-i™ Blastocyst Vitrification kit. Blastocysts were not collapsed for vitrification. Warming of the blastocysts was performed on the morning of the scheduled frozen embryo transfer and the embryos cultured for 2-3 hours prior to transfer to the patient. Warming was performed with the Vitrolife Rapid-i™ Blastocyst Thawing kit and the blastocysts were cultured in COOK® Sydney IVF Blastocyst culture medium until the time of transfer.

Results

Fresh embryo transfers

A total of 299 fresh embryo transfers were included in the

analysis: 53 patients had an embryo transfer of 2 embryos on Day 5 with no blastocysts, 33 patients had a Day 5 embryo transfer with 1 blastocyst and 1 other embryo that had not formed a blastocyst, the remaining 213 patients had an embryo transfer with 2 blastocysts. Transfer data was excluded for those patients from which the origin of the implanting embryo could not be determined, for example; one gestation from a double embryo transfer containing two embryos, only one of which has formed a blastocyst. For a twin gestation, the data was included.

The clinical pregnancy rate for those patients who had no blastocysts transferred was 38%. This pregnancy rate was not significantly different from the pregnancy rate of the group where the transfer pool contained at least one blastocyst (42%), however, the pregnancy rate from the group with two blastocysts transferred was significantly different (56%) than the other two groups.

The implantation rate for the group with no blastocysts transferred was significantly different from the implantation rate for the group with two blastocysts transferred (25% versus 38% respectively). The multiple pregnancy rate for the group with no blastocysts transferred (16% of pregnancies) was significantly different from the other two groups (29% and 30% of pregnancies respectively).

Embryo Transfers with warmed blastocysts

For the time period of the analysis, 228 embryo transfers were included in the analysis: 117 of the transfers were performed using warmed Day 5 vitrified blastocysts, and

Figure 1. Embryos classifiedasblastocystsatthetimeofpatientembryo transfer.

Figure 2. Embryos classified as “not blastocysts” on Day 5.Embryos middle and right have a small cavitation forming, however, the distinct inner cell mass and trophectoderm of the blastocyst are not yet distinguishable.


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111 transfers were warmed Day 6 vitrified blastocysts. The Day 5 embryo transfer group had an average of 1.5 embryos transferred and the Day 6 group an average number of 1.4 embryos. The clinical pregnancy rate was not significantly different for the Day 5 vitrified blastocysts versus the Day 6 vitrified blastocysts (56% versus 45%). There were also no significant differences found in the implantation rate for each group (45% versus 41% respectively).

Discussion

This study examined whether or not slowly developing blastocysts were suitable for embryo transfer and Day 6 vitrification by using the outcome measure of clinical pregnancy. The data in Figure 3 illustrate that the group of patients who had an embryo transfer, of two embryos, on Day 5 with no blastocysts had a significantly different clinical pregnancy rate (38%) than those patients with two

blastocysts transferred on Day 5 (56%). If the Day 5 embryo transfer pool contained one blastocyst and one slower developing embryo, the clinical pregnancy rate did not increase (42%).

The impact of at least one blastocyst in the embryo transfer pool was seen in the multiple pregnancy rates for each group. The multiple pregnancy rate in the two groups with blastocysts were significantly higher than the group without any blastocysts transferred (29% and 30% versus 16% respectively). The inclusion of an extra embryo in the transfer pool, whether or not it is at the blastocyst stage, does not significantly increase the pregnancy rate, in the case of the one blastocyst and one slower embryo, but significantly increases the multiple pregnancy rate. Therefore, caution must be exercised in any transfer where at least one blastocyst is being transferred. A second, even if it is a slower growing embryo, will significantly increase multiple pregnancies.

Vitrified and warmed blastocysts do not seem to exhibit a difference in the clinical pregnancy rates or implantation rates no matter what the day of blastocyst development (Day 5 or Day 6). This data is in agreement with a study published by El-Toukhy et al who also found that there was no difference in the results between the clinical pregnancies for frozen-thawed Day 5 or Day 6 blastocysts.9 Other groups have found significant differences between the pregnancy results depending upon the original day of blastocyst freezing.10,11 Differences in culture media, culture conditions or diversity in patient ovarian stimulation protocols may account for differences seen between clinics different groups and may affect the inherent quality of the embryos.

Further studies would be required in order to explain the differences between the slower growing embryo that is transferred fresh and the slower growing embryo that is vitrified on Day 6. One possible explanation is that the slower embryo, when transferred fresh, may miss the window of implantation due to the further embryo development that is required while in the uterus before this embryo is ready to implant.

The blastocyst that forms on Day 6 is vitrified at the same stage of development as the blastocyst vitrified on Day 5, albeit one day later. The Day 5 and Day 6 blastocyst, when warmed, become equalized respective to the endometrial lining of the patient because they are thawed and transferred at the same time. The slower growing embryo has now become better synchronized with the uterine lining due to the extra day of growth in culture prior to vitrification. A prospective randomized study would be required to determine if this hypothesis is correct.

Composition of embryo transfer pool

(%)

a

a

a

a

b

b

b

ba

Figure 3. Pregnancy results for patients receiving fresh embryos for transfer on Day 5. For each measure, bars with the same letter(a,b)arenotsignificantlydifferent.Differentlettersrepresentsignificantdifferences(p<0.05) by Chi-square test.

n=117

n=111 n=173n=154

(%)

Figure 4. PregnancyresultsforvitrifiedandwarmedblastocystsfrozenonDay5orDay6.NosignificantdifferencesfoundbyChi-square analysis.


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Conclusions

Although this study was based upon data collected retrospectively, it does illustrate that even for patients with no blastocysts forming by the time of fresh Day 5 embryo transfer, the pregnancy rate, although significantly lower, remains acceptable. After the completion of the transfer, any excess embryos grown to Day 6 for vitrification will, upon thawing, have the same pregnancy rate as those blastocysts vitrified on Day 5. Therefore embryos developing slower than the established timelines should not be excluded from consideration due to the acceptable pregnancy and implantation rates.

References

1. MacKenna A, Crosby J, Zegers-Hochschild F. Sibling blastocyst development as a prognostic factor for the outcome of day-3 embryo transfer. Reproductive Biomedicine Online, 2013:26:486-490

2. Seli E, Gardner D, Schoolcraft W, et al. Extent of Nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertility and Sterility, 2004: 82(2):378-383

3. Tao T, Robichaud A, Mercier J, et al. Influence of group culture strategies on the blastocyst development and pregnancy outcome. J Assist Reprod Genet, 2013:30:63-68

4. Never A, Zintz M, Stecher A, et al. The impact of paternal factors on cleavage stage and blastocyst development analyzed by time-lapse imaging – a retrospective observational study. J Assist Reprod Genet, 2015:32(11):1607-14

5. De Cassia Savio Figueria R, Souza Setti A, Braga D, et al. Blastocyst morphology holds clues concerning the chromosomal status of the embryo. International Journal of Fertility and Sterility, 2015:9(2):215-220

6. Nasiri N, Eftekhari-Yazdi P. An overview of the available methods for morphological scoring of per-implantation embryos in In Vitro fertilization. Cell Journal, 2015:16(4):392-405

7. De Munck N, Santos-Ribeiro S, Mateizel I, et al. Reduced blastocyst formation in reduced culture volume. J Assist Reprod Genet, 2015:32(9):1365-70

8. Wdowiak A, Bakalczuk S, Bakalczuk G. The effect of sperm DNA fragmentation on the dynamics of the embryonic development in intracytoplasmic sperm injection. Reprod Biol, 2015:15(2):94-100

9. El-Toukhy T, Wharf E, Walavalkar R, et al. Delayed blastocyst development does not influence the outcome of frozen-thawed transfer cycles. BJOG, 2011:118:1551-6

10. Shapiro B, Richter K, Harris D, et al. A comparison of day 5 and day 6 blastocyst transfers. Fertility and Sterility, 2001:75:1126-30

11. Levens E, Whitcomb B, Hennessy S, et al. Blastocyst development rate impacts outcome in cryopreserved blastocyst transfer cycles. Fertility and Sterility, 2008:90(6):2138-2143

global® DMSO Blastocyst Vitrification Kit global® DMSO Blastocyst Warming Kit

Easy-To-Use and a High Performance Systemglobal® DMSO Blastocyst Vitrification


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During meiosis and mitosis, chromosomes segregation is a critical event taking place in the meiotic or mitotic spindle, which has been proven

to be a thermo-sensitive structure. Changes occurring in temperature during oocyte or embryo manipulation may irreversibly affect spindle integrity, resulting in chromosomal abnormalities. Therefore, maintaining a stable temperature around 37°C during all IVF procedures is essential to obtain successful results.

In order to improve knowledge and understanding of Quality Control and Assurance in the IVF lab, we focused our attention on temperature fluctuations of culture dishes during manipulation. We performed several studies to show how different factors in the lab can affect the temperature of the microdroplets of medium, as would be used for the culture of embryos and gametes.

Materials and Methods

We first examined temperature change when taking the dishes outside of the incubator and placing them on a heated laminar flow hood. The variables we took into account were: type of oil used (low vs. high viscosity oil, LiteOil® and LifeGuard® respectively, LifeGlobal®), culture medium microdroplets distribution (periphery vs. center of the dish), type of culture dish (Nunc 35mm and 60mm vs. embryo GPS® and µdrop GPS® dishes) and temperature set-point of heated surfaces (37°C vs. 40°C).

In the second experiment, we mimicked real working conditions by placing dishes back into a regular or benchtop incubator and compared temperature recovery timings. We used previously prepared and heated dishes, and allowed them to cool on a warm surface until a temperature of 34.0°C (±0.5°C) inside the microdroplets was achieved. Dishes were then placed back into either a regular incubator (HeraCell 150i) or a benchtop incubator (K-MINC®, COOK), and measurements were taken every 30 seconds until a stable temperature was reached within a 30 min period.

Nunc and embryo GPS® dishes were prepared with 50µl culture medium microdroplets overlaid with 14ml of oil in 60mm dishes and 5ml of oil in 35mm dishes. In µdrop GPS® dishes, two different kinds of set-up were compared: 20µl microdroplets were covered with 3ml or with 14ml of

oil. These two strategies could help us understand how the volume of oil interferes in temperature maintenance. (Fig.1)

Temperature measurements were performed inside the microdroplets using a fine gauge thermocouple probe (accuracy +/- 0.01°C, TC, Okolab®), with readings every 30 seconds. All measurements were performed in duplicate, and a maximum difference of 0.5°C was accepted between replicates. At all times, the temperature of the heated surfaces and the incubators was externally controlled using small temperature loggers (accuracy +/- 0.07°C, iButtons®, Thermodata®) to ensure temperature stability during the readings.

Results

Temperature loss

Different set-up conditions resulted in greatly varying temperature fluctuations. Only few of the tested combinations were successful in holding temperature inside an optimal range between 36 and 37.9°C for more than 10 minutes. When placed on a 40°C surface, Nunc dishes were stable regardless of the dish size, type of oil used or microdroplet distribution. On a 37°C surface, embryo GPS® dishes prepared with low viscosity oil and µdrop GPS® dishes prepared with high viscosity oil were the most efficient in maintaining an optimal temperature.

Other combinations resulted in excessively warm (>38°C) or cold (<35°C) temperatures within a short period of time. Overall, high viscosity oil was better able to resist cooling than low viscosity oil. Temperature loss was more rapid in a peripheral microdroplet distribution than in a central distribution. A 40°C heated surface set-point prevented an early cooling of the dishes, but in some cases resulted in overheating of culture medium microdroplets, especially in dishes prepared with low viscosity oil. GPS® dishes were the most efficient in resisting temperature loss outside the incubator. (Fig. 2)

How to assure a stable temperature in culture medium microdroplets?by Enric Mestres, Ivette Vanrell, Maria Garcia,Nuno Costa-Borges, Gloria Calderón

Embryotools SL, Parc Científic de Barcelona,Av. Doctor Marañon 8, 08028 Barcelona (Spain)

You can contact Enric Mestres and Ivette Vanrellat [email protected]

iVeTTe Vanrell

enric MiSTreS


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Temperature recovery

There were no major differences in temperature recovery between types of dishes, microdroplets distributions or high and low viscosity oils. However, there was a considerable variation in temperature recovery between regular and benchtop incubators. Whereas it took approximately 17 minutes for the temperature inside the microdroplets to reach 36.5°C in a regular incubator, it took only 6 minutes for dishes in a benchtop incubator to reach the same temperature. Even though temperature inside all the incubators was 37°C, microdroplets never reached that temperature when using a regular incubator. On the contrary, microdroplets reached and stabilized at 37°C within 10 minutes of being placed back inside a benchtop incubator. (Fig. 3)

Discussion and conclusions

The results of these studies show that many factors related to the set-up of culture dishes, culture system, and conditions in the IVF lab can directly affect heat transfer in culture medium microdroplets. Depending on the settings, disposables, and incubators used, temperature may behave differently and irreversibly compromise gametes and embryo development and quality.

Although temperature fluctuations are inevitable during embryo manipulation in routine IVF tasks, the optimization of some working factors may greatly reduce this impact both outside and inside the incubators. Seemingly small factors such as the type of oil and plastic dishes used, distribution of the culture medium microdroplets within the dish, or the set-point of the heated stages can have a big impact on

temperature fluctuations outside of the incubator. The oil acts as a temperature buffer which delays cooling.

Central microdroplets have a bigger surface contact with oil than peripheral microdroplets, which are in direct contact with the walls of the dish. Therefore, central microdroplets do not lose temperature as fast as peripheral ones.

Low viscosity oil is more easily influenced by external temperature than high viscosity oil, resulting in faster temperature loss and recovery. Microdroplets covered with low viscosity oil lose temperature faster if placed on a 37°C surface, but can become overheated when placed on a 40°C surface. Oil volume also has an impact in temperature loss and recovery. Larger volumes of oil delay temperature loss, but also result in slower temperature recovery.

GPS® dishes are specially designed to be more efficient in temperature gathering since the bottom of the dish is in direct contact with the warmed surface. On the contrary, Nunc dishes have a little but determining protruding edge around the bottom that prevents from direct warming.

As we can see, carefully choosing the type of incubators used in the laboratory seems essential, as regular incubators will need much more time to warm up dishes than benchtop incubators. Accordingly, an excess of door openings in regular incubators can induce temperature to get out of range for longer periods of time.

In conclusion, keeping simple details controlled in the lab can minimize stress on embryos and gametes, avoiding unnecessary risks during embryo manipulation. However, the unavoidable differences in conditions between IVF laboratories make it necessary for each lab to study and optimize their working protocols.

Figure 1.1. 60mm culture dishes with 50µl droplets covered with 14ml of oil. A) Peripheric distribution in NUNC dish. B) Central distribution in NUNC dish. C) embryo GPS® dish.

Figure 1.2. 35mm culture dishes. A) Peripheric distribution in NUNC dish with 50µl droplets covered with 5ml of oil. B) Central distribution in NUNC dish with 50µl droplets covered with 5ml of oil. C) µDrop GPS® dish with 20µl droplets covered with 3ml of oil. D) µDrop GPS® dish with 20µl droplets covered with 14ml of oil.

Figure 1. Dishes and microdroplets distributions used


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Figure 2. Temperature loss

Figure 3. Temperature recovery


Universal GPS®

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4-Well GPS® DishKey Features and Big Benefits

• 4 large wells with GPS bottoms for easy viewing and manipulation • GPS bottom for embryo settling in the center of the wells for quick location of

the embryo • Wells are designed to reduce shadows and have concise focal positioning • 4 large wells will hold up to 300 microliters of media and oil • 4 smaller outer wells hold up to 100 microliter of media and oil • Wide well design allows quick viewing and oil overlay of each well as well as

the entire dish • No pyrogenics, clean medical grade • CE and ISO Registered, FDA 510(k) Cleared • 1-cell MEA and LAL Tested

embryo GPS®

μDrop GPS®

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• Holding oocytes prior to ICSI • Large Volume Culture and Group Culture • Assisted Hatching using laser • Denuding before ICSI • Cryopreservation and Thawing

4-Well GPS®


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global® Blastocyst Fast Freeze®

Easy-To-Use and a High Performance System

global® Blastocyst Fast Freeze® Kit Easy three-step Fast Freeze®

global® Blastocyst Fast Freeze® Thawing Kit

Does not require special Vitrification deviceand uses regular sealable straws.

“We perform the majority of our embryo cryopreservation at the blastocyst stage and have found the global® Blastocyst Fast Freeze® system to be the simplest, most efficacious and cost-effective method we have tried. As a result, it has been in use exclusively in our laboratory for over a year and a half and the survival rate upon warming is in excess of 99% with stellar pregnancy rates. That the system employs 0.25 cc straws and larger volumes made it extremely simple to learn for the entire technical staff and has resulted in dramatic cost reductions since more expensive cryopreservation devices are not required. Additionally, it has been the perfect cryopreservation adjunct to trophectoderm biopsy and provides consistently high survival and pregnancy rates in conjunction with PGS/PGD. I have recommended the system to a number of my colleagues; none have been disappointed.” Dr. Thomas Pool, PhD of The Fertility Center of San Antonio, [email protected](No commercial ties with this company.)

Each kit includes 1 sleeve of 10 Universal GPS® dishes.


CONFERENCES

ESHRE 2015 – Lisbon, Portugal, July 2015

ASRM 2015 – Baltimore, Maryland, October 2015

Conferences and Workshops


CONFERENCES

India Conference – February 2016Embryolab Academy Workshop in

Leuven, Belgium – March 2016

Attended by LifeGlobal® Group

ALPHA 2016 – Copenhagen, Denmark, May 2016

Bulgaria Workshop - May 2016


CONFERENCES

American Society for Reproductive Medicine

Scaling New Heights in Reproductive Medicine

October 15-19, 2016Salt Lake City, Utah

www.asrm.org

ART in the Era of Personalized Medicine

September 14-16, 2016Toronto, Ontario, CANADA

www.cfas.ca


CONFERENCES

www.pcrsonline.org

Welcome to PCRS 2017 Annual MeetingMarch 22 - 26, 2017

Advances in Reproductive Medicine

ESHRE 2017

European Society of Human Reproduction and Embryology

33rd Annual Meeting

July 2-5, 2017Geneva, SWITZERLAND

www.eshre.eu


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Over 15 Years of Published Clinical ResultsProven ConsistencyProven Performance> 150 Independent Published Clinical Studies

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global® global®for Fertilization

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LG PGDBiopsy Medium

LiteOil® LifeGuard® Oil

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