according to the literature on the worldwide prevalence of involuntary childlessness between 52.6 to 72.4 million couples can benefit from some form of medical intervention to achieve a pregnancy  ( mascarenhas et al . , 2012 ) , most of them being residents of developing countries or resource - poor countries  ( rutstein and iqbal , 2004 ; boivin et al . , 2007 ) . its implications are strongly related to and mediated by different socio - cultural factors . especially in developing countries , most societies are organized such that children are necessary for care and maintenance of the older parents . moreover , in the absence of social security systems , older people are economically completely dependent on their children  ( dyer and patel , 2012 ) . consequently , the inability to bear children is a tragedy and childlessness can be considered as an important and neglected reproductive health problem in developing countries  ( bergstrom , 1992 ; leek et al . , 1993 ; fathalla et al . , 2006 ; van balen and bos , 2009 ; gerrits and shaw , 2010 ; dhont , 2011a ) . bilateral tubal occlusion due to sexually transmitted diseases and pregnancy - related infections is the most common cause of infertility in dc and is only treatable by using ivf related procedures which are either unavailable or too costly  ( ombelet et al . , 2008 ) . during the past 35 years of ivf this should be regarded as a great success story if a large part of the world population could benefit from these new technologies , which is currently not the case in most developing and even developed countries . to optimise infertility care in terms of availability , affordability and effectiveness the simplification of ivf procedures is urgently needed . as part of the walking egg project  ( dhont , 2011b ; ombelet , 2013 ; 2014 ) and based on previous findings and experience  ( van blerkom and manes , 1974 ; swain , 2011 ) we developed a new simplified method of ivf culturing , called the we lab method . with this new system , specifically designed for low resource settings , we can avoid the complexity of the use of medical gases , high - tech incubation equipment and infrastructure typical of ivf laboratories in high resource settings . in this closed system we only used 1000 - 5000 motile washed spermatozoa for insemination of the eggs , with very promising results , which makes this technique usable for a large part of the actual ivf / icsi population , even if a moderate or severe male factor is involved  ( van blerkom et al . , 2014 ) . since development from insemination to transfer is undisturbed and in the same tube until embryo transfer , we can avoid many problems frequently occurring in regular ivf laboratories , such as unwanted temperature changes , air quality problems etc . the birth of seven healthy babies from this simplified ivf system was recently reported  ( van blerkom et al . , 2014 ) . in this paper we describe the pregnancy outcome and birth of another four healthy babies born after cryo - thawing and embryo - transfer of embryos resulting from this we lab system . a simple chemical reaction was used to produce co2 de novo , namely the combination of a weak base  ( sodium bicarbonate ) , a weak acid  ( citric acid ) and water . the result is a defined atmosphere that is stable for prolonged periods such that culture medium could be equilibrated in advance of an ivf cycle . consequently the possibility of changes in temperature , atmosphere or ph that could have adverse developmental effects are minimized or eliminated . we only need a uniform temperature which can be maintained quite simply and without the need for conventional and more expensive incubators  ( fig .  1 ) . pre - selection of the embryo s to be used for transfer can be done without opening the vacutainers . the we lab system is a closed system and intended to enable fertilization and embryo development to occur undisturbed in the same tube until day 3 , when uterine transfers are done  ( fig . 1 ) . the patients described in this paper are part of a prospective non - inferiority study performed at the zol hospitals in genk , belgium . the methodology of this study was described before  ( van blerkom et al . , 2014 ) . couples enrolled in this study required ivf as assessed by the standard protocol of infertility evaluation used at zol hospitals . in all patients it was the first ivf attempt , all women were below 36 years of age and only ivf cycles with at least 8 oocytes recovered at follicular aspiration were included . participation in this clinical trial required informed consent documentation and this study was approved by the local ethical committee of genk and the ethical committee of the free university of brussels , and registered as b.u.n . according to the belgian law for this specific patient population , single embryo transfer  ( set ) was obliged  ( ombelet et al . , 2005 ) . therefore a reasonable number of embryos , resulting from the we lab simplified ivf system , were cryopreserved . surplus embryos were cryopreserved using a controlled rate programmable freezer  ( planer kryo 360 1.7 , t.s . briefly , embryos were exposed for 10 minutes to each of the freezing solutions with increasing concentrations of proh , supplemented with human serum albumin  ( up to 12mg / ml ) and gentamicin  ( 0.01  mg / ml ) . solution one was equilibrated to 37c in air whilst solutions 2 and 3 were used at room temperature . the embryos were loaded into a 0.3ml cbstm high security embryo straw  ( cryobiosystem ) and both ends of the straw were sealed . the straws were places into the controlled rate programmed freezing machine  ( precooled to 20c ) and kept at 20c for 10 minutes . the straws were cooled to -6c at a rate of -2c per minute , held at this temperature for 4  minutes and then seeded manually and held at -6c for another 15 minutes . it was then cooled to -30c at a rate of -0 , 3c per minute and subsequently cooled to -160c at a rate of -10c per minute and held there for 10 minutes . the straws were plunged in liquid nitrogen and stored in liquid nitrogen vessels . for thawing of the frozen embryos , the straws were removed from the liquid nitrogen vessel and warmed to 30c for 1 minute . the cryoprotectant was removed by transferring the embryos to stepwise dilutions of the thawing solutions from the sydney ivf thawing kit sits-5000 at room temperature . the embryos were warmed to 37c , evaluated on their survival and cleavage stage and cultured in embryo culture medium . a simple chemical reaction was used to produce co2 de novo , namely the combination of a weak base  ( sodium bicarbonate ) , a weak acid  ( citric acid ) and water . the result is a defined atmosphere that is stable for prolonged periods such that culture medium could be equilibrated in advance of an ivf cycle . consequently the possibility of changes in temperature , atmosphere or ph that could have adverse developmental effects are minimized or eliminated . we only need a uniform temperature which can be maintained quite simply and without the need for conventional and more expensive incubators  ( fig .  1 ) . pre - selection of the embryo s to be used for transfer can be done without opening the vacutainers . the we lab system is a closed system and intended to enable fertilization and embryo development to occur undisturbed in the same tube until day 3 , when uterine transfers are done  ( fig . the patients described in this paper are part of a prospective non - inferiority study performed at the zol hospitals in genk , belgium . the methodology of this study was described before  ( van blerkom et al . , 2014 ) . couples enrolled in this study required ivf as assessed by the standard protocol of infertility evaluation used at zol hospitals . in all patients it was the first ivf attempt , all women were below 36 years of age and only ivf cycles with at least 8 oocytes recovered at follicular aspiration were included . participation in this clinical trial required informed consent documentation and this study was approved by the local ethical committee of genk and the ethical committee of the free university of brussels , and registered as b.u.n . according to the belgian law for this specific patient population , single embryo transfer  ( set ) was obliged  ( ombelet et al . , 2005 ) . therefore a reasonable number of embryos , resulting from the we lab simplified ivf system , were cryopreserved . surplus embryos were cryopreserved using a controlled rate programmable freezer  ( planer kryo 360 1.7 , t.s . scientific , perkasie , pa , usa ) and sydney ivf cryopreservation kit sics-5000 . briefly , embryos were exposed for 10 minutes to each of the freezing solutions with increasing concentrations of proh , supplemented with human serum albumin  ( up to 12mg / ml ) and gentamicin  ( 0.01  mg / ml ) . solution one was equilibrated to 37c in air whilst solutions 2 and 3 were used at room temperature . the embryos were loaded into a 0.3ml cbstm high security embryo straw  ( cryobiosystem ) and both ends of the straw were sealed . the straws were places into the controlled rate programmed freezing machine  ( precooled to 20c ) and kept at 20c for 10 minutes . the straws were cooled to -6c at a rate of -2c per minute , held at this temperature for 4  minutes and then seeded manually and held at -6c for another 15 minutes . it was then cooled to -30c at a rate of -0 , 3c per minute and subsequently cooled to -160c at a rate of -10c per minute and held there for 10 minutes . the straws were plunged in liquid nitrogen and stored in liquid nitrogen vessels . for thawing of the frozen embryos , the straws were removed from the liquid nitrogen vessel and warmed to 30c for 1 minute . the cryoprotectant was removed by transferring the embryos to stepwise dilutions of the thawing solutions from the sydney ivf thawing kit sits-5000 at room temperature . the embryos were warmed to 37c , evaluated on their survival and cleavage stage and cultured in embryo culture medium . the outcome of three pregnancies resulting in four live births are described in figure  2 . the first couple  ( case  1 ) was referred to our infertility clinic with a history of primary infertility for more than two years . the couple agreed to be included in the prospective study in which our simplified ivf culture system  ( scs ) was compared with regular ivf culture system  ( rcs ) , described by van blerkom et al . ( 2014 ) . in the first  ( fresh ) ivf attempt 14 oocytes were retrieved , a single embryo transfer  ( set ) from scs was performed according to the protocol of the study  ( figure  1a ) . four embryos were cryopreserved and two months later one embryo originating from the scs was thawed and transferred in a cc  ( clomiphene citrate ) stimulated cycle . the patient became pregnant and delivered at 41  weeks of gestation of a boy weighing 4060  grams . the second couple  ( case  2 ) had a history of primary infertility for 26 months . a moderate male factor could be detected , an investigation showed no female abnormalities . the couple also volunteered to be included in the prospective study , as mentioned above . 13 oocytes were retrieved and a set from scs was performed , without success  ( fig . 2b ) . a few months later a set was performed after thawing of two embryos , one embryo did nt survive . she became pregnant and delivered at 39 1/2 weeks of gestation of a boy weighing 4100 grams . the third couple  ( case 3 ) had a history of primary infertility for 28  months . a moderate male factor could be detected , an investigation of the women revealed the diagnosis of anovulation due to pcos  ( polycystic ovary syndrome ) . the couple agreed to be included in the prospective study and 19 oocytes were retrieved during the first ivf attempt . because of a high risk for ohss  ( ovarian hyperstimulation syndrome ) it was decided to trigger ovulation with an agonist  ( decapeptyl 0.2  mg i m , ipsen , belgium ) instead of using hcg  ( pregnyl 5000 , msd , belgium ) . all embryos  ( n  =  9 ) were cryopreserved  ( figure  2c ) . a few months later a det  ( double embryo transfer ) was performed after thawing of three embryos originating of the rcs , without success . during the second attempt the gestation was uneventful , she delivered at 38  weeks of gestation of a boy weighing 3170 grams and a girl of 2900  grams . infertility is a universal health issue and the large majority of childless couples are residents of developing countries . it is a silent population of more than 50 million couples facing the consequences of childlessness day by day . these consequences of involuntary childlessness are usually much more dramatic in developing countries and can create broader problems compared to western societies , particularly for women  ( van balen and gerrits , 2001 ; dyer et al . , 2004 ; van balen and bos , 2009 ; dhont , 2011a ) . despite its relatively high prevalence and the cultural values associated with childbearing , infertility remains a low priority area for local health care providers and community leaders  ( ombelet et al . most health care providers argue that the limited recourses should only be given to programs focussing on reducing sexually transmitted diseases  ( stds ) , postpartum and post abortion complications rather than offering high - technology treatments to infertile couples , the so - called limited resources argument . severe male infertility due to stds and female infertility due to tubal block can only be treated by  expensive  assisted reproductive technologies  ( art ) which are not available at all or only within reach of those who can afford it , mostly in a private setting . lowering the costs associated with ivf laboratory procedures , namely fertilization and culture of eggs and embryos , was the first aim of the walking egg project  ( ombelet , 2013 ; 2014 ) . in a recent paper we described that using a very simplified and inexpensive method of ivf culturing good results can be obtained in terms of clinical pregnancy rate per cycle and the birth of healthy babies  ( van blerkom et al . , 2014 ) . the results with the simplified ivf method were comparable with the outcome results after regular high - tech ivf . the next question might be : do the surplus embryos obtained with this new method survive cryopreservation and thawing with reasonable outcome results . in this paper we report the birth of four healthy babies , the result of two singletons and one twin pregnancy . our preliminary outcome results in fresh ivf cycles and after cryopreservation and thawing of surplus embryos clearly show that this simplified system may make advanced infertility treatment in dc practical , affordable and effective . if the results of the initial study can be reproduced in other centres we believe that the simplified we lab procedure will open a new era in the history of ivf as the method not only offers affordable and successful access to ivf , it will make assisted reproductive techniques available to a much larger part of the worlds infertile population , an important breakthrough in terms of human rights , equity and social justice for infertile couples in resource - poor countries  ( johnson et al . , 2014 ; , 2006 ; pennings et al . , 2009 ; vayena et al . , 2009 ; ombelet , 2011 ; dhont , 2013 ) .
background : infertility care is one of the most neglected health care issues in developing countries  ( dc ) , affecting more than 50 million couples . the social stigma of childlessness still leads to isolation and abandonment . bilateral tubal occlusion due to sexually transmitted diseases and pregnancy - related infections 
 is the most common cause of infertility in dc . 
 consequently most cases of infertility are only treatable by using assisted reproductive technologies which are either unavailable or too costly . 
 lowering the laboratory costs associated with ivf is a crucial step to make ivf affordable for a larger part of the world population . 
 we recently developed and described a new simplified method of ivf culturing , called the twe lab method . 
 our initial results in fresh ivf cycles showed that ivf methodology can be significantly simplified and result in successful outcomes at levels that compare favourably to those obtained in high resource programs.case-reports : we report three pregnancies and four live births as a result of transferring five cryo / thawing embryos which were developed after using the simplified twe lab system . 
 the two singleton babies delivered vaginally , for the twin pregnancy a caesarean section was performed . 
 all babies were healthy , the perinatal outcome was uneventful in all cases.conclusion : we provide proof - of - principle evidence that transferring cryopreserved / thawed embryos obtained with our twe lab simplified culture system can lead to successful pregnancies and healthy live births .