Optimising embryo development in a protective in vitro environment.

The G5 Series. Optimising embryo development in a protective in vitro environment. The G5 Series. Helping nature succeed.

The next generation of culture media ART evolved. Since the introduction of the very successful GI Series, continued research has resulted in improved culture conditions, now available as the G5 Series. With the G5 Series, culture media takes another significant step forward. Oxidative stress and how to counteract it - vitroshield The presence of reactive oxygen species, ROS or free oxygen radicals, has major implications in embryo culture. The unique addition of vitroshield to the G5 Series culture system will help to protect against ROS. Embryo development will thereby be less vulnerable in vitro. ph corresponds with embryo intra-cellular conditions Read more on page 5 Control and regulation of intra-cellular ph (phi) is essential for normal embryo development and maintenance of viability. In the G5 Series, ph has been lowered to be close to the embryo physiological level. Read more on page 7 Even in the best environment, you still need some protection against the surroundings. 2 The G5 Series Vitrolife 1.080527.2 INTL

Increased protection against infection Infection during embryo culture is a potential risk in laboratories around the world. To protect from possible infections, the broad spectrum antibiotic gentamicin is included in the G5 Series. Photo: Getty Images/Taxi Cover photo: Vitrolife G5 Optimising embryo development The compositions of the products in the G5 Series are built on the in vivo conditions of the female reproductive tract, with the nutrients and energy sources that gametes and embryos require. This provides a supportive environment during the entire culture period from oocyte to transfer at the cleavage or blastoyst stage. Read more on page 9 Easy to use With the G5 Series, IVF procedures have become easy. Only four products are required, G-MOPS for all manipulations at ambient atmosphere, G-IVF for sperm preparations and fertilisation, G-1 for cleavage stage embryo culture and G-2 for culture of blastocysts. Read more on page 11 Excellent clinical data With the G5 Series, clinics have been able to obtain high pregnancy and implantation rates. Read more on page 12 Hyaluronan for increased viability Hyaluronan is an important component of the G5 Series that enhances embryo viability, and cryosurvivability, and facilitates embryo implantation. Read more on page 8 Vitrolife 1.080527.2 INTL The G5 Series

What is the G5 Series? The G5 Series is a new generation culture media system designed to improve success rates while providing a new kind of protection against detrimental environmental factors. The unique features of the G5 Series will allow your embryos to develop in a more protective and supportive environment than ever before. Based on the successful GI Series. Fewer products compared with former series. A common set of ions to prevent intracellular stress induced by moving embryos through different types of solutions. Gradients of carbohydrates are present to meet the different requirements of the embryo at successive stages of development. Amino acids are included according to the needs of the embryo. Prior to compaction, non-essential amino acids and methionine reduce homeostatic stress, while post-compaction essential amino acids stimulate inner cell mass development. Why G5? Improved embryo development In vitro environmental protection Improved cryotolerance Easy to use Built on proven success Protect your embryos. The G5 Series Vitrolife 1.080527.2 INTL

vitroshield for embryo protection. With the G5 Series you can protect embryo culture from in vitro environmental hazards. vitroshield is the combination of hyaluronan, gentamicin and the antioxidant lipoate in the culture system. Hyaluronan enhances embryo viability and cryosurvivability. Gentamicin offers broad spectrum protection against bacterial agents. Lipoate is one of the most potent antioxidants known; it protects against reactive oxygen species present in the culture environment. ph close to the intra-cellular level protects from perturbations in embryo development. What is oxidative stress? Oxidative stress is caused by free reactive oxygen species, so called ROS. Examples of ROS are the superoxide (O 2 - ) and hydroxyl (OH ) radicals, and hydrogen peroxide (H 2 O 2 ). What are the sources of ROS and what damage can they cause in ART? There may be multiple sources of ROS in an IVF setting. One possible source of ROS is visible light. Light is able to induce ROS production and cellular damage such as oxidation of bases and DNA strand breaks; an exposure of > 5 min is sufficient to cause a major increase in H 2 O 2 in mouse embryos. 1 ROS may be responsible for causing increased embryo fragmentation resulting through increasing apoptosis. A major contributor to the production of ROS is the concentration of oxygen itself. Typically, clinics tend to use ambient oxygen, around 20%. This is in stark contrast to the levels found within the female reproductive tract, closer to 5%. Therefore, under typical IVF laboratory conditions, there is an excess of oxygen, culminating in increased oxidative stress. The vitroshield was developed with such stress in mind. How can oxidative stress be overcome in ART? Oxidative stress can be overcome by reducing the generation of ROS directly. Fertilisation and embryo development in vivo occur in an environment of low oxygen tension and without exposure to light. During ART, it is important to avoid conditions that promote ROS generation and expose gametes and embryos to ROS. During culture, low oxygen tension is more effective at improving implantation and pregnancy rates than higher oxygen tension. IVF culture is constantly exposed to free oxygen radicals and pollutants originating from light, air and environment. Alternatively, one can increase the amounts of antioxidants available. It has been demonstrated that the antioxidant ascorbate can alleviate the oxidative stresses associated with slow freezing. 2 Now it has been determined that the inclusion of lipoate in the culture media can help to alleviate the negative impact of oxygen on embryo development, as manifest by more 8-cell embryos and blastocysts developing. What are antioxidants? An antioxidant has been defined as any substance that, when present at a low concentration, significantly delays or inhibits oxidation of an oxidizable substrate. Vitrolife 1.080527.2 INTL The G5 Series 5

Comparison of embryo development in sub-optimal culture dishes: the protective effect of the G5 Series. The dishes had failed the mouse embryo assay three times. Seventy-two embryos were cultured to the blastocyst stage (96 h) in each group. From these data, it appears that G5 media confer some form of protection to the developing embryo in culture, as well as protecting against oxidative stress. The nature of the embryo toxin has not been identified. Blastocysts Blastocysts Hatching Cell Day 4 Day 5 Day 5 Number GI Series 44.8 71. 26.7 2.3 G5 Series 63.5 ** 85.6 *.8 ** 68.3 * Significantly different from GI Series ; *, P<0.05; **, P<0.01 Lipoate improves embryo development in 20% O 2 (CF1 mouse). Percent 100 75 50 25 0 Ref: Vitrolife, data on file. Control Lipoate Blast dev ** Cell no Under normal conditions, antioxidants convert ROS to H 2 O. There are two major types of antioxidants in the human body: enzymatic antioxidants and nonenzymatic antioxidants. Enzymatic antioxidants are also known as natural antioxidants; they neutralize excessive ROS and prevent them from damaging the cellular structure. Non-enzymatic antioxidants are also known as synthetic antioxidants or dietary supplements, (e.g., vitamins). Which antioxidants have been used in IVF culture media? Amino acids, pyruvate, glutathione and vitamins (e.g., C and E) added to the IVF media have antioxidant properties. However, in their reduced forms vitamin C and glutathione are unstable in solution and become ineffective. Furthermore, vitamin E is insoluble in an aqueous phase. 3, 4 What is Lipoate? Lipoate (alpha-lipoic acid or 6,8-dithio octanoic acid) is a potent antioxidant. It is thought to be synthesised in the mitochondria of animals, bacteria and plants. It contains soluble properties allowing interactions in both aqueous and lipid phases, a unique feature among antioxidants. Furthermore, lipoate is capable of regenerating other antioxidants including glutathione. For this reason it has been called the antioxidant of antioxidants. 5 vitroshield may also provide a protection against toxic compounds originating from contact supplies. Following the development of the G5 Series culture media formulations, it was observed during testing that the media appeared to possess the ability to provide additional protection to embryos from certain embryo-toxic substances. It was determined that when culture dishes that had previously failed the 1-cell mouse embryo assay (performed using a simple medium lacking amino acids and albumin) were used to culture embryos in either GI or G5 Series media, the new Series 5 supported higher rates of embryo development. 6 Lipoate is one of the most potent antioxidants known; it protects against reactive oxygen species present in the culture environment. vitroshield protects your embryos in culture from ph induced stress, free oxygen radicals, infections and cryodamage. + + + = LIPOATE gentamicin hyaluronan The G5 Series Vitrolife 1.080527.2 INTL

ph close to the intra-cellular level protects from perturbations in embryo development. Control and regulation of intracellular ph (phi) is essential for normal embryo development and maintenance of viability The ability to regulate ionic homeostasis is essential for normal cell development. Intracellular levels of protons together with calcium, magnesium and phosphate regulate a multitude of cellular functions such as cell division, differentiation, cytoskeletal dynamics and metabolism. Aberrations in cellular homeostasis result in perturbed cell function and loss of developmental competence. This has significant implications for the IVF laboratory, whose role is to maintain embryo viability. Therefore, the IVF laboratory must ensure that cellular ionic stress is minimized. 7 Levels of phi and its regulation in embryos The resting level of phi in human embryos seems to be between 7.1 and 7.2 8. Cells have two mechanisms to regulate phi: (1) short term regulation and (2) long term regulation. Short term regulation Intracellular intrinsic buffering is the physiochemical and organelle buffering of protons and is the non-co 2 contribution to minimizing changes in ph caused by changes in proton concentrations. This intrinsic buffering capacity may be a mechanism to compensate for the reduced capacity to restore ph because of lack of functional membrane transporters in the oocyte. Short term regulation works within seconds. Long term regulation This is achieved by specific transport systems in the cell membrane. However, these transporters can take several minutes to restore phi to physiological level. Acidosis is relieved by the Na + /H + antiporter that is found in nucleated cells. Alkalosis is regulated by the HCO 3- / Cl - exchanger. The HCO 3- / Cl - exchanger transports bicarbonate out of the cell in exchange for Cl - and regulates phi in the 9, 10 neutral to alkaline range. Intracellular ph regulation by oocytes In contrast to embryos, mammalian oocytes appear to lack any transport mechanisms for regulating phi 8, 11. Activity of the transport system appears gradually until maximum activity is reached around 8 to 10 hours after egg activation. In vivo, the mammalian oocyte and early embryo are surrounded by cumulus, which persists for several hours after fertilisation. Cumulus cells are surrounded by a matrix of glycoproteins and glycoaminoglycans such as hyaluronan. It is therefore postulated that in vivo, the oocyte and early embryo are protected by the cumulus and are not required to regulate phi until after the transport system is initiated. Intracellular ph and control of embryo metabolism The mammalian cleavage stage embryo relies on oxidation of carboxylic acids and amino acids for energy. Glucose is not used as an energy source until after Schematic diagram of known mechanisms for the regulation of intracellular levels of ph. The Na + /H + antiporter is used by embryos to regulate the phi against an acid challenge. The HCO 3 - /Cl - exchanger is used by embryos to regulate phi against an alkaline challenge. the late four-cell stage 12. Intracellular ph is a powerful regulator of many key enzyme pathways and reactions that control both oxidative metabolism and glycolysis. Increases in phi above the physiological level of 7.1 7.2 are known to increase the activity of the key regulatory enzyme in glycolysis, phosphofructokinase (PFK). Only small increases in PFK activity can cause disproportionately large increases in glycolytic pathway activity. The activity of PFK can be significantly altered by increasing ph only by 0.2 units. Such activation of glycolysis could be associated with developmental arrest in culture. Therefore, changes in phi have been shown to have a significant effect on essential cellular processes such as regulation of cytoskeletons as well as the ability of the embryo to maintain adequate levels of energy production. Thus, it appears that regulation of phi is central to the ability of the preimplantation embryo to regulate enzyme activity and therefore metabolism. Vitrolife 1.080527.2 INTL The G5 Series 7

Photo: Martin Barraud/Photographer s choice Hyaluronan for increased viability. Improves embryo cryosurvivability Supports preimplantation embryo development in culture Facilitates embryo implantation The inclusion of Hyaluronan in the G5 Series Hyaluronan is the major glycosaminoglycan present in follicular, oviductal and uterine fluids. 13, 14, 15 It is a unique macromolecule which is found in tissues and body fluids in every mammalian species. As a physiological substance, hyaluronan is rich in the female reproductive tract and also abundant around oocytes, especially during the periovulation period. Following 16, 17 studies in mammalian species, hyaluronan, along with recombinant human albumin, not only support human embryo development during the preimplantation stages in culture, but also facilitate the implantation of human embryos. 18 It has also been demonstrated that hyaluronan and recombinant human albumin can significantly improve embryo 19, 20 cryosurvivability. Improved results after cryopreservation when embryos have been cultured and transferred in hyaluronan-containing media. Embryos from 293 patients were included. All embryos were cultured in GI Series media and transferred on day 2. 21 8 The G5 Series Vitrolife 1.080527.2 INTL Clinical pregnancy rate 40 Percent 30 20 10 0 22.7 32.9 G-2 ver 3 EmbryoGlue Transfer media Implantation rate Percent 40 30 26.3 20 10 17.7 0 G-2 ver 3 EmbryoGlue Transfer media

Optimising embryo development. Learning from the mother and the embryo. The need for sequential media Physiological conditions in the female tract vary from the oviduct to the uterus. Therefore, in order to meet the changing requirements of the developing embryo in culture, sequential media were developed. Sequential media have been proven to be highly effective in supporting the development of viable human embryos and blastocysts. Scientific background Learning from the mother The physiological conditions in the female reproductive tract, for example the concentrations of pyruvate, lactate, glucose, amino acids and oxygen, change from the ampulla of the oviduct to the uterus in a gradient manner. In the ampulla the pyruvate and lactate concentrations are at their highest point and decrease down the reproductive tract to reach their lowest point in the uterus. The opposite is true for glucose. The glucose concentration is low in the ampulla and high in the uterus. 22 These nutrient gradients have a significant impact on embryo physiology and viability. The concentration of the gases oxygen and carbon dioxide are different in the oviduct compared to in the uterus, 23, 24, 25 which in turn reflects ph. The intracellular ph (phi) of embryos from mammalian species including the human is around 7.2 8 and it appears that external ph (pho) does not regulate phi as much as initially thought. Rather, embryos have the capacity to regulate phi in a culture environment of ph 7.2 to 7.4. When embryos are cultured in a ph too different from phi the rate of development is decreased. Amino acids increase the intracellular buffering capacity of the embryo, and are therefore key regulators of phi. Those amino acids that buffer phi are present in high concentrations in the female reproductive tract 26 and are present in G media. Hyaluronan is present in the reproductive tract and the highest concentration is found in the uterus. Hyaluronan appears to assist in sperm selection during fertilisation, promote embryonic cell proliferation and facilitate embryo implantation. Learning from the embryo The embryo s requirements differ with developmental stage. The pre-compacted embryo has a low metabolic activity. It has a limited capacity to utilise glucose and it generates energy from low levels of oxidation of pyruvate/lactate and amino acids. Cell proliferation and compaction are stimulated by the presence of non-essential amino acids 27, 28 and EDTA. 2001 Terese Winslow Vitrolife 1.080527.2 INTL The G5 Series 9

Photo: Lennart Nilsson Clinical data showing improved embryo development with the G5 Series compared to the GI Series. Oocytes from 26 patients were split between the GI Series and the G5 Series culture systems. Development to the 8-cell and blastocyst stages were significantly improved with the G5 Series. Media system Mean no 8-cell embryos Blastocysts of 2PN on day 3 on day 5 GI Series 7± 0.5 55.0 % 50.8 % G5 Series 7± 0.5 72.4 % ** 61.6 % * Significantly different from GI Series; * P<0.05; ** P<0.01 6 In contrast, the embryo post-compaction has a high metabolic activity. It uses glucose as the preferred nutrient and requires a wider array of amino acids for cell proliferation and differentiation, as well as specific vitamins to maintain oxidation. Amino acids have several key roles in embryo development. They function as biosynthetic precursors and energy substrates. Additionally, they act as regulators of energy metabolism, as osmolytes, antioxidants and chelators. More specifically, non-essential amino acids and glutamine (those amino acids abundant in the oviduct) increase cleavage rates of the zygote and increase viability of the cleavage stage embryo as well as stimulate trophectoderm development. In contrast, those amino acids required by somatic cells (essential amino acids) appear to confer no benefit to the cleavage stage embryo and can actually impair viability. However, post 8-cell, essential amino acids stimulate inner cell mass development and increase fetal development after blastocyst 29, 30, 31 transfer. The presence of protein (albumin) is also important to the embryo. Albumin works as a ph buffer, colloid osmotic regulator and as carrier of growth factors. Albumin also plays a role as a scavenger, and as a surfactant making manipulation of gametes and embryos possible. Ammonium Consistent with Vitrolife's ongoing commitment to safety, all G media contain the heat stable dipeptide alanylglutamine, rather than free glutamine, in order to greatly reduce the buildup of embryo-toxic ammonium. Clinical data has shown that ammonium is detrimental to the development of the human embryo. 32 The G5 Series family members G-RINSE Solution for rinsing of contact materials and for washing of the cervix. Not for culture. No protein supplementation needed. Contains the same ionic backbone as the other products in the G5 Series. Ready to use. 10 The G5 Series Vitrolife 1.080527.2 INTL G-MOPS / G-MOPS PLUS * Medium for handling and manipulating oocytes and embryos in ambient atmosphere. Contain lipoate, non-essential amino acids and MOPS buffer. For use at ambient atmosphere. No equilibration in CO 2 needed. No protein supplementation needed for oocyte retrieval. For ICSI and embryo manipulation use G-MOPS PLUS or G-MOPS supplemented with G-MM or HSA-solution. G-GAMETE * Medium for handling and manipulating oocytes and embryos in ambient atmosphere. Contains HSA and non-essential amino acids. Ready to use. G-IVF / G-IVF PLUS * Medium for preparation and handling of gametes and for in vitro fertilisation. Contain taurine, citrate, fructose and non-essential amino acids. G-1 / G-1 PLUS * Medium for culture of embryos from the pronucleate stage to day 2 or 3. Contain lipoate, hyaluronan, citrate, taurine, methionine and non-essential amino acids. G-2 / G-2 PLUS * Medium for culture of embryos from day 3 to the blastocyst stage.

Easy to use. of the G5 Series products: One medium for semen preparation and fertilisation G-IVF. One medium for the ICSI procedure G-MOPS. A handling medium that you can incubate with CO 2 G-GAMETE. All products designated PLUS are supplemented with HSA-solution and ready to use. All products (except G-GAMETE and EmbryoGlue ) are also available without albumin. All media contain gentamicin as an antibacterial agent. Contain citrate and both essential, non-essential amino acids and a high concentration of hyaluronan. EmbryoGlue Implantation promoting medium for the embryo transfer procedure. Contains a high concentration of hyaluronan and recombinant human albumin for safety. Both essential and non-essential amino acids are included. G-PGD Medium for embryo biopsy. Free of calcium and magnesium. MOPS buffered for use at ambient atmosphere. Contains both essential and non-essential amino acids. HSA-solution * Medium supplement. Contains 100 mg human serum albumin/ml. To be added to the G5 Series products not designated PLUS. Strictly controlled pharmaceutical human serum albumin. G-MM Medium supplement. Contains 50 mg recombinant human albumin/ml. To be added to the G5 Series products not designated PLUS. G-FreezeKit Blast Media for freezing of blastocyst stage embryos. MOPS buffered for use at ambient atmosphere. Contains both essential and non-essential amino acids. G-ThawKit Blast Media for thawing of blastocyst stage embryos. MOPS buffered for use at ambient atmosphere. Contains both essential and non-essential amino acids. * CAUTION. All blood products should be treated as potentially infectious. Source material from which this product was derived was found negative when tested in accordance with current FDA required tests, HIV, types 1 and 2; HBV; HCV, and HTLV types I and II. No known test methods can offer assurance that products derived from human blood will not transmit infectious agents. Vitrolife 1.080527.2 INTL The G5 Series 11

Photo: Longview/Image Bank Building on proven success. Scientific results supporting the G-Series. Improved results with hyaluronan-containing G-Series media confirmed in clinical studies. Clinical comparison GIII Series versus G5 Series The Center for Reproduction at Uppsala University Hospital, Sweden, has been using the G- Series media for embryo culture and handling for several years. Below is a retrospective comparison of their results with the GIII and G5 Series. In addition, the clinics results on cryopreservation cycles are shown. Material and methods The GIII Series was used from January until September 14, 2007. The G5 Series was used from September 15 to the end of the year. The two G-Series media were used for embryo handling and culture Results Fresh cycles GIII G5 transfers GIII Series transfers G5 Series Total total Jan mid Sept total Mid Sept Dec No of oocyte retrievals 70 292 178 SET DET SET DET No of embryos transferred 534 326 204 122 208 122 86 No of embryo transfers 0 265 204 61 165 122 43 Pregnancy rate/transfer 7% 46% 45% 48% 49% 53% 37% Ongoing pregnancy rate/transfer 33% 32% 31% 34% 35% 36% 30% Implantation rate 27% 31% 21% 36% 22% Cryo cycles Total SET DET No of embryo transfers 07 224 83 No of embryos transferred 390 224 166 Pregnancy rate /transfer 5% 34% 42% Ongoing pregnancy rate/transfer 27% 25% 31% Implantation rate 23% 25% 23% 12 The G5 Series Vitrolife 1.080527.2 INTL

but not for sperm preparation. Embryo transfers were performed on day 2 or 3, either in G-1 ver 3 PLUS or in G-2 v5 PLUS. EmbryoGlue was used for all transfers of cryopreserved embryos. The majority of transfers are single embryo transfers (SET), transfer of two embryos (DET) is applied only in older patients and in patients with several previously failed cycles. Conclusion The results show that the G5 Series performs equal to, or better, than the GIII Series on embryo transfers of cleavage stage embryos. The data for both fresh cycles and for cryocycles also show that a high pregnancy rate can be maintained even if only one embryo is transferred. REF: F Hambiliki. Center for Reproduction, Uppsala University Hospital, Uppsala, Sweden. Data on file 2008. 33 Multicenter evaluation of the G5 Series - media for handling and culture of human embryos The G5 Series media for handling and culture of human embryos was introduced at ESHRE, July 2007. In order to verify that the products give the anticipated outcome, a clinical evaluation was initiated. Material and Methods The evaluation started in August 2007 and ended in December 2007. 15 clinics from 9 countries (Belgium, Canada, Denmark, Finland, Germany, Sweden, Turkey, United Kingdom and USA) participated. 1356 embryos from 585 patients were included in the evaluation. The majority of embryo transfers were performed on day 3 (134 transfers on day 2, 394 transfers on day 3 and 56 transfers on day 5). The complete media system was used for each Series, including OVOIL. The G5 Series consists of G-MOPS/ G-MOPS PLUS or G-GAMETE, G-IVF/ G-IVF PLUS, G-1/ G-1 PLUS, G-2/ G-2 PLUS. The responsible embryologists were asked to work according to the G5 Series manual. Data was collected using a Case Report Form (CRF) provided by Vitrolife. During culture of embryos, scoring was performed each day according to a protocol (included in the CRF). Embryo development was assessed in the morning no later than 10 am. Results Clinical pregnancy rate (CPR) and implantation rate (IR) increased when embryos were transferred at a later developmental stage, reaching the highest level of 59 % CPR for blastocysts transfers, see graph. Clinical pregnancy rates and implantation rate according to embryo transfer day Percent 60 50 40 30 20 10 0 37 CPR IR 27 49 26 Conclusion The above data confirm that the G5 Series perform well in clinical settings around the world for transfers on day 2, day 3 and day 5. REF: Vitrolife. Data on file. 2008. 59 Day 2 Day 3 Day 5 35 Fertilisation rates improved comparison G5 Series with GIII Series The former version of the G-series media by Vitrolife, the GIII Series, contained two different media for sperm preparation and fertilisation, respectively, G-SPERM and G-FERT. With the new G5 Series, only one medium, G-IVF, is needed for both applications. With the aim to evaluate if G-IVF performs better than G-SPERM and G-FERT, a clinical test was performed. Material and methods The media were tested for both ICSI and conventional IVF. For ICSI cases, the media were used for sperm preparation and holding during the time between oocyte retrieval and injection. For conventional IVF, the media were used for sperm preparation and for insemination, usually over night. That means that for conventional IVF cases, the oocytes were subjected to the media for a much longer period of time than for ICSI cases. The hypothesis was therefore that a greater difference, if any, between old and new media would be seen for conventional IVF cases. G-IVF was tested on all together 5390 oocytes and G-SPERM + G-FERT on 5796 oocytes. 15 clinics in 9 countries participated. Results As can be seen in the graph below, for conventional IVF cases, the fertilisation rate was significantly improved when G-IVF of the new G5 Series was used, compared with the media of the GIII Series. Fig 1. Fertilisation results 80 *** 60 69 58 Percent 40 20 0 75 76 IVF ICSI Fertilization rate between the G-IVF group (G5 Series, blue bars) and the G-SPERM +G-FERT group (GIII Series, grey bars) divided in IVF and ICSI. (G5 IVF; n=626 and GIII IVF; n=860, G5 ICSI; n=4764 and GIII ICSI; n=4936) *** p< 0.001 G5 compare to GIII OR 1.6; 95th confidence interval (CI) 1.3 1.9, Chi-square with Yates correction. Conclusion The use of G-IVF of the G5 Series resulted in a significantly higher fertilisation rate compared to the GIII Series products for the same application. As discussed earlier, it was postulated that if Vitrolife 1.080527.2 INTL The G5 Series 13

the new G-IVF medium would perform better than the old products, it would have the greatest impact on conventional IVF cases. The clinical evaluation confirmed this hypothesis. REF: Vitrolife. Data on file. 2008 Improved embryo development with the G5 Series comparison between G5 and GIII Series Background Before the introduction of the G5 Series at ESHRE 2007, the new products were clinically evaluated. The result from the clinic with the highest number of participating patients is found below. Material and methods A total of 337 treatment cycles were included. Patients were allocated to have their embryos cultured either in the GIII Series or in the G5 Series. ICSI was performed on all cases. The embryos (n=1600) were cultured to either the 8-cell stage or to the blastocyst stage (n=295). There were no significant differences in duration or cause of infertility or mean age between the two patient groups. Results More good quality embryos were available for transfer and cryopreservation after culture in the G5 Series on both day 3 and day 5. The number of embryos with 8 cells on day 3, were significantly Embryo development in GIII and G5 Series 60 50 55 58 * 40 30 37 42 Percent 20 10 0 Good quality embryos 8 cells day 3 * p<0.05 Comparison G5 and GIII Series Day 3 embryo transfer Day 5 embryo transfer G5 GIII G5 GIII No. of cycles 147 149 21 20 No. of embryo transfers (%) 141(95.9) 140 (93.9) 21 20 2PN fertilisation (%) 787 (76.2) 813 (75.7) 148 (76.6) 147 (75.7) Grade 1 & 2 embryos day 3 (%) 9 (58.4) 441 (55.1) 92 (63.4) 88 (60.6) 8 cell embryos (%) (43.2)* 303 (37.8) 59 (40.6) 54 (37.2) Grade 1 & 2 embryos for day 3 transfer (%) 246 (62.2) 232 (57.1) Good quality blastocysts for transfer day 5 (%) 28 (62.2) 27 (61.3) Implantation rate % 25.3 23.1 40.0 38.6 Cycles with embryos for cryo (%) 52 (36.8) 45 (32.1) 5 (23.8) 4 (20.0) * p<0.05 higher in the G5 Series compared to in the GIII Series. Conclusion The use of the G5 Series resulted in a significantly improved embryo development. In addition, implantation rate and number of blastocysts on day 5 were higher in the G5 Series compared to the GIII Series. Due to the low number of embryos, these parameters did not reach statistical significance. REF: Balaban B et al, American Hospital of Istanbul, Turkey. Data on file 2007. 34 Enhanced cryosurvival following culture in hyaluronancontaining G-Series media Background Hyaluronan in embryo culture media has a beneficial effect on cryosurvival and subsequent implantation rate in a number of mammalian species. The aim of this study was to investigate if embryos cryopreserved after culture in hyaluronan-containing media had a better survival rate and implantation rate than human embryos cultured in media without hyaluronan. Material and methods A total of 126 frozen embryo transfer cycles were performed after a failed fresh transfer. G-media version 2 without hyaluronan or G-Series version I media with hyaluronan were used for embryo culture. Embryos were cryopreserved on day 3. To investigate further development after thawing, the embryos were cultured for approximately 24 hours prior to transfer. Results Frozen embryo transfers were performed in 64 cycles (G-Series version III) and 62 cycles (G-Series version 2). Mean number of thawed embryos and cryosurvival rates were similar between groups. Of the thawed day 3 embryos, 58.3% in the G-Series I group and 26.2% in the G-Series 2 group reached the morula stage (P<0.01). Implantation rate per transferred embryo was significantly higher when in vitro culture was undertaken in G-Series version I media with hyaluronan prior to cryopreservation. Embryo development after cryopreservation and transfer 100 Percent 80 60 40 20 0 Cryosurvival Embryo dev to morulae * p<0.05; ** p<0.01 Conclusion Hyaluronan in embryo culture media increases embryo viability resulting in improved development post thawing and higher implantation rate after embryo transfer. REF: Balaban B and Urman B. RBMOnline 2005; Vol 10 No 4 pp485-491 35 ** G-Series 2 G Series * Implantation 14 The G5 Series Vitrolife 1.080527.2 INTL

References 1. Goto et al. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radical Bio. Med. 1993; 15 69-75. 2. Lane et al. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. Human Reproduction. 2002; 17: 2693. 3. Guerin P, El Mouatassim S and Menezo Y. Oxidative stress and protection against reactive oxygen species in the preimplantation embryo and its surroundings. Hum Reprod Update. 2001; 7(2):175-189. 4. Agarwal A, Gupta A and Sharma R. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005; 14(3): 28. 5. Bilska A and Wlodek L. Lipoic acid the drug of the future? Pharmacol Rep. 2005; 57(5):570-577. 6. Gardner DK and Larman M. Fertility Laboratories of Colorado, data on file. 2007. 7. Lane M and Gardner D. Regulation of ionic homeostasis by mammalian embryos. Seminars in Reprod Med 2000; 18 (2). 8. Phillips L et al. Intracellular ph regulation in human preimplantation embryos. 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In: Gametes: Development and Function. Eds A Lauria et al. Serono Symposia; Roma. p 239-272. 16. Gardner DK et al. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Hum Reprod. 1999; 14(10):2575-2580. 17. Stojkovic M et al. Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro. Reproduction. 2002; 124(1):141-153. 18. Schoolcraft W et al. Increased hyaluronan concentration in the embryo transfer medium results in a significant increase in human embryo implantation rate. ASRM; 2002; Seattle, USA. 19. Gardner DK, Maybach J and Lane M. Hyaluronan and RHSA increase blastocyst cryosurvival. Proc 17th World Congress on Fertility and Sterility; 2001; Melbourne. p 226. 20. Lane M et al. 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Fusion Design & Advertising, Sweden G5 Series. Helping nature succeed. Optimising embryo development in a protective in vitro environment. 510(k) clearance. G-RINSE, G-MOPS, G-MOPS PLUS, G-GAMETE, G-IVF, G-IVF PLUS, G-1 v5, G-1 v5 PLUS, G-2 v5, G-2 v5 PLUS, EmbryoGlue, G-PGD, G-MM, G-FreezeKit Blast and G-ThawKit Blast have CE mark. Patent No. US 6,838,235, Patent No US 6,762,053, Patent No. NZ523481. Other patents pending. Vitrolife Sweden AB, Faktorvägen 13, SE-434 37 Kungsbacka, Sweden. Tel +46-31-721 80 00, Fax +46-31-721 80 90, fertility@vitrolife.com, www.vitrolife.com Vitrolife, Inc., 3601 South Inca Street, Englewood, Colorado 80110, USA, Tel 866-VITRO US (866-848-7687), Fax 866-VITROFAX (866-848-7632), fertility.us@vitrolife.com This brochure contains information regarding various tests and clinical trials relating to Vitrolife products. This information on tests and clinical trials relating to Vitrolife products is only a summary provided for information purposes about Vitrolife products. The information is provided as is without any warranties, expressed or implied, including but not limited to the implied warranties of suitability or eligibility for a particular purpose and/or success of treatment on an individual basis. Products and information may have changed since the printing of this brochure. For more information see www.vitrolife.com. Vitrolife 1.080527.2 INTL