We now have more than a decade of experience with time-lapse microscopy in the clinical in vitro fertilization (IVF) setting, gaining considerable insights into the value of knowing not just the developmental status of an embryo at a specific time, but also how and when it got there. Numerous studies have also defined some types of embryonic bad behavior that are not apparent from classic static morphologic evaluations, events such as reverse cleavage and direct cleavage. But it is the acquisition and application of morphokinetic data to embryo selection as a tool for enhancing IVF outcomes that has generated the widest interest. In a comprehensive multiyear examination of the influence of time-lapse on outcomes, Zaninovic et al. (1Zaninovic N. Zhan Q. Rosenwaks Z. Time-lapse implementation in a clinical setting: outcome results.in: Meseguer M. Time-lapse microscopy in in-vitro fertilization. Cambridge University Press, Cambridge, UK2016: 144-164Google Scholar) reported significant increases in clinical pregnancy, ongoing pregnancy, implantation, and live birth rates with the use of a time-lapse incubator compared with a standard incubator. There has been some discussion if it is the undisturbed environment of the time-lapse incubator, not the use of morphokinetic data, that yields better-quality embryos and is therefore responsible for the improved clinical results; however, work from several laboratories refute this. For example, Kirkegaard et al. (2Kirkegaard K. Hindkjaer J.J. Grondahl M.L. Kesmodel U.S. Ingerslev H.J. A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator.J Assist Reprod Genet. 2012; 29: 565-572Crossref PubMed Scopus (116) Google Scholar) saw no difference in the number of 4-cell embryos on day 2, 7–8-cell embryos on day 3, blastocysts on day 5, and both clinical pregnancy and implantation rates when time-lapse incubation was compared with conventional incubation. In general, morphokinetic analysis of human embryogenesis has shown that embryos showing quicker development produce better results on transfer than those developing more slowly. The all-consuming issue for the experimentalist is to ferret out which parameters carry significant value in adjusting outcome, and a host of algorithms have been proposed and revised over the past decade to meet this goal. In this vein, dal Canto et al. (3dal Canto M. Bartolacci A. Turchi D. Pignataro D. Lain M. de Ponti E. et al.Faster fertilization and cleavage kinetics reflect competence to achieve a live birth after intracytoplasmic sperm injection but this association fades with maternal age.Fertil Steril. 2021; 115: 665-672Abstract Full Text Full Text PDF Scopus (10) Google Scholar) evaluated 1,390 embryos retrospectively, scoring several early morphokinetic parameters following intracytoplasmic sperm injection, including time to pronuclear fading (tPNf) and time to reach the 2-cell (t2), 3-cell, 4-cell , 5-cell, and 8-cell stages. In the total population of patients, embryos producing live birth on transfer reached all stages earlier than those that did not. When patients were stratified by maternal age, the relationship held true for those under 37 years but not so for those 37 and older. The exception to this was t2, which was independent from maternal age in its association with live birth. That morphokinetic timings can vary with age is not a new suggestion, but it is demonstrated in this work with data taken from specific early parameters with the use of a large number of embryos of known outcome. It is intriguing to consider the biological basis for the fading of this relationship with maternal age. Because early blastomeres have not fully evolved the cell cycle gaps and checkpoints of the mature somatic cell, the possibilities are more limited but still exceedingly complex. To enter mitosis requires activation of maturation-promoting factor, the complex of cycle-dependent kinase 1 (Cdk-1) and cyclin B. This kinase activity is normally inhibited by phosphorylation of Cdk-1 by Wee1 and activated by dephosphorylation from CDC25 activity. Hörmanseder et al. (4Hörmanseder E. Tischer T. Mayer T.U. Modulation of cell cycle control during oocyte-to-embryo transitions.EMBO J. 2013; 32: 2191-2203Crossref PubMed Scopus (36) Google Scholar) suggest that the timing of Cdk-1 activation and inactivation can be adjusted by the modulation of regulatory circuits acting on Cdk-1/cyclin B. Suffice it to say it is complicated and involves synthesis and degradation of cyclin B, ubiquitin-mediated cyclin B proteolysis, E3 ubiquitin ligase anaphase-promoting complex/cyclosome, CDC20, or Cdh-1 plus a plethora of phosphorylation events. Another perplexing element is why is the parameter t2 immune from the age-associated fading of a relationship to pregnancy seen in other early parameters? We have much to learn. The findings reported by dal Canto et al. (3dal Canto M. Bartolacci A. Turchi D. Pignataro D. Lain M. de Ponti E. et al.Faster fertilization and cleavage kinetics reflect competence to achieve a live birth after intracytoplasmic sperm injection but this association fades with maternal age.Fertil Steril. 2021; 115: 665-672Abstract Full Text Full Text PDF Scopus (10) Google Scholar) make one thing very clear: One cannot ignore a discussion of maternal age when counseling patients about the potential value of morphokinetics. Also, for those patients that have spent an hour or two on the internet and now equate their understanding of human embryology with yours, advise them to keep reading—some things that are true one day may not be so the next. Faster fertilization and cleavage kinetics reflect competence to achieve a live birth after intracytoplasmic sperm injection, but this association fades with maternal ageFertility and SterilityVol. 115Issue 3PreviewTo assess the relationship of early developmental kinetics with competence to provide a live birth and the impact of maternal age in this context. Full-Text PDF

There are certain steps in a process that are essential to successful outcome but, taken alone, are not at all predictive that success will occur. For example, one cannot deny that the winner of the Kentucky Derby got on a horse, an essential act, just as the winner of the Indianapolis 500 got into a car, equally essential. However, the isolated acts of getting on a horse or into a car are not predictive of one winning those respective races. There are other issues, to say the least. Nonetheless, in embryology, we tend to use the presence of a polar body in an aspirated oocyte as evidence that it is mature and, thus, destined for ensuing fertilization and embryonic development.

The purpose of this chapter is to review some of the factors that influence laboratory and clinical outcomes, broadly under the heading of optimal handling techniques. Two principal environments are encountered – inside and outside the incubator. The chapter will address media buffers, gas atmosphere, timing and setting up culture or holding vessels, protection of medium performance, temperature relative to handling gametes and embryos, lighting, pH, incubation choices, and workflow. The importance of the interplay between these variables cannot be overlooked.

It has long been established that the successful operation of an IVF laboratory requires dedicated quality control and assurance, implemented at the appropriate level (Mortimer and Mortimer, 2005Mortimer D. Mortimer S. Quality and Risk Management in the IVF Laboratory. Cambridge University Press, Cambridge, UK2005Google Scholar). In recent years we have witnessed increasing complexity within the clinical IVF laboratory, and, along with it an increasing demand on the laboratory staff to ensure optimal functioning of equipment and provisions to provide proper environmental conditions for gamete and embryo culture, striving to maximise the chances of conception and live birth. A persistent question remains in many practitioners’ minds, however: What is an appropriate level of monitoring, and what is (are) the best way(s) to achieve it? The paper by Palmer et al., 2019Palmer G.A. et al., A comparison among 36 assisted reproduction laboratories monitoring the environmental conditions and instrument parameters using the same quality control application. 2019.In this issue.Google Scholar in this issue of RBMO has gone some way to address this issue by comparing quality control data from 36 clinics across 12 countries, all of which used a cloud-based application for quality control monitoring. It was observed that there is heterogeneity in practices among laboratories, and, interestingly, that investment in quality control was substantial in countries in which accreditation of laboratories is required, while it was minimal in almost half of the laboratories surveyed. It is evident that the reason we know comparatively little about quality control practices in assisted reproductive technology (ART) laboratories worldwide is that most of the data are buried in binders full of checklists and forms that at one time adorned clip boards or were taped to incubators, refrigerators and the like in the lab. The time and effort required for collation and analysis of data in this format has always been considerable, and usually beyond what is feasible for most clinics. As a result, although data are collected, they are rarely examined in detail apart from a monthly cursory review and acknowledgement. Thus, the development of a cloud-based application to collect, store, retrieve and analyse quality control data may be considered a natural progression. This is a logical and very practical approach, valuable to large and small clinics alike. Cloud-based quality management is standard in many mature industries such as aeronautical, automotive, pharmaceutical, food, and others; these are businesses that use quality control as a proactive and effective tool for improving performance. The report by Palmer et al., 2019Palmer G.A. et al., A comparison among 36 assisted reproduction laboratories monitoring the environmental conditions and instrument parameters using the same quality control application. 2019.In this issue.Google Scholar is in effect one of the first attempts to equip IVF laboratories with the technologies used elsewhere to great effect. This work therefore represents a novel approach and a 'call-to-action', though much remains to be done. The call for standardization is logical but are we ready to define those standards? Although we now have tools that will allow for such an analysis, as yet we have not tied instrument performance to outcomes in a comprehensive manner, in a way that would make standardization evidence-based. Indeed, the report by Palmer does not relate any of the measured parameters to clinical outcome. There remain a number of basic questions regarding quality control practices, specifically, whether we are measuring the correct parameters. This is especially perplexing given that there are at least 200 variables that can impact IVF outcome (Pool et al., 2012Pool T.B. Schoolfield J. Han D. Human embryo culture medium comparisons.in: Smith G.D. Swain J.E. Pool T.B. Embryo Culture, Methods and Protocols. Methods in Molecular Biology 912. Humana press,, New York,2012: 367-386Google Scholar). Moreover, one could ask, are we taking measurements at appropriate frequencies? Are our measurement intervals catching important events like freezer cycling? Do we accept indirect measures, such as incubator CO2, instead of biologically meaningful ones such as pH? We require further proof from transfer outcomes that temperature ranges must be narrow. Without this, we could be 'overdoing it' without meaningfully contributing to better outcomes; this could amount to wasting valuable laboratory resources. Hence, an advantage that emerges from the use of such a cloud-based system is that data sharing may assist in the fine-tuning of such ranges and help to optimise those parameters linked to clinical outcome. Further studies are required to help ascertain key parameters and the frequencies of monitoring, which will vary depending on the parameter in question. In its classical usage, quality control in the IVF laboratory is simply documentation to show that equipment and instruments are functioning and doing so within a predetermined range of operational parameters tied to the analytical phase of a testing event. That proof is then archived either physically in binders or electronically on a server where it remains dormant, unless retrieved for accreditation purposes. Cloud storage and tools to rapidly deposit and retrieve quality control data for any instrument over any specified time interval with simply a few keystrokes changes all of that. The timely discovery of faulty equipment/instrumentation followed by immediate corrective actions through effective quality management practices is the gold standard of quality control; however, the potential linking of quality control performance to enhanced patient care and outcomes without opening a binder or spreadsheet suggests that the cloud may truly possess a silver lining.

If we have gleaned any one general principle from the time-lapse literature with regard to embryo selection, it is this: successful outcome from the transfer of a blastocyst depends somewhat upon it reaching that stage but even more so upon how and when it was reached. The simple visualization of an emerging fluid-filled space in a morula belies the underlying workings of a myriad of ordered molecular interactions. Without a doubt, blastulation is a complex process, with the resulting blastocyst representing the successful derivation of divergent cell lineages, a culmination of an ordered sequence of events that begins with the “start signal,” compaction, in mammalian embryos. Reviewed elegantly by Sozen et al. (1Sozen B. Can A. Demir N. Cell fate regulation during preimplantation development: a view of adhesion-linked molecular interactions.Dev Biol. 2014; 395: 73-83Crossref PubMed Scopus (32) Google Scholar), this process begins as junctional complexes form at apicolateral and lateral sites on blastomeres, followed by polarization within the outer cells. From mouse studies, it is clear that cell polarity proteins (Par 1, Par 3, aPKC, Jam 1, Ezrin) along with transcription factors, such as Cdx2, play critical mechanistic roles in this process. In fact, mouse knockout studies reveal a complex genomic interplay, indicating that both maternal and zygotic Cdx2 expression are required for blastocyst formation from morulae (2Jedrusik A. Cox A. Wicher K. Glover D. Zernika-Goetz M. Maternal-zygotic knockout reveals a critical role of Cdx2 in the morula to blastocyst transition.Dev Biol. 2015; 398: 147-152Crossref PubMed Scopus (36) Google Scholar). Similar processes are present in human embryogenesis as well, but blastocyst formation from the initiation of compaction is an inefficient process, at least in contemporary culture systems, and one that either slows or even fails on a somewhat regular basis. For example, Iwata et al. (3Iwata K. Yumoto K. Sugishim M. Mizoguchi C. Kai Y. Iba Y. et al.Analysis of compaction initiation in human embryos by using time-lapse cinematography.J Assist Reprod Genet. 2014; 31: 421-426Crossref PubMed Scopus (41) Google Scholar) used time-lapse cinematography to observe thawed human embryos, cryopreserved at early stages, and saw that compaction was initiated at the eight-cell stage in 86.1% of embryos. Of these, only approximately half developed into good-quality blastocysts. If compaction began earlier, as it did in 13.9% of embryos, the results were even worse, with only 18.8% forming good-quality blastocysts. Determining why this happens is not easy, even with tools for the dynamic study of early embryogenesis, such as time-lapse microscopy. As Kirkegaard et al. have shown (4Kirkegaard K. Sundvall L. Erlandsen M. Hindkjaer J.J. Knudsen U.B. Ingerslev H.J. Timing of human preimplantation embryonic development is confounded by embryo origin.Hum Reprod. 2016; 31: 324-331PubMed Google Scholar), variation in the temporal events of development is complicated, in that it is most likely caused by a combination of factors, not a single one, and is highly dependent upon patient factors; thus, embryo origin is a major confounder in the timing of preimplantation embryogenesis. As an example, they note that the morphokinetic parameters of time to initial blastocoel formation and time to full blastocoel formation are both affected by FSH dose and by the number of prior IVF attempts. Patient age was also seen to influence blastulation: time to initial blastocoel formation occurred significantly later with older age. Just how extrinsic factors impinge upon intrinsic mechanisms governing blastocoel formation remains a mystery. Perhaps with time our understanding of the requirements for the successful determination and differentiation of trophectoderm and inner cell mass lineages will allow for intervention should they falter—after all, why not “precision embryology”? As it is now, however, we are faced with decisions regarding what to do with slowly developing embryos, ones that often are all that a patient has available after IVF. What to do—discard, fresh transfer, culture further, culture further and transfer, culture further then vitrify? This is the dilemma addressed in the current issue of the journal by Haas et al. (5Haas J. Meriano J. Bassil R. Barzilay E. Zilberberg E. Casper R.F. Developmental potential of slow-developing embryos: day-5 morulae compared with day-5 cavitating morulae.Fertil Steril. 2019; 111: 105-111Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), who examined the developmental fate of delayed morulae, ones forming on day 5 vs. cavitating morulae formed on day 5. How significant an event is cavitation to further development and viability if embryogenesis is delayed? As the authors demonstrate in this retrospective analysis of a large number of delayed embryos, it depends and is not necessarily all that intuitive. Stopping short with a delayed embryo on day 5 when another 24 or even 48 hours of culture time is available for the embryo to continue development is not the likely strategy of many assisted reproductive technology programs. However, Haas et al. (5Haas J. Meriano J. Bassil R. Barzilay E. Zilberberg E. Casper R.F. Developmental potential of slow-developing embryos: day-5 morulae compared with day-5 cavitating morulae.Fertil Steril. 2019; 111: 105-111Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) compared the pregnancy rates obtained from day-5 fresh transfer between delayed morulae and cavitating delayed morulae, seeing no statistical difference. Yet when each of these was cultured further until day 6 for potential vitrification, the delayed morulae produced significantly fewer blastocysts, only one in five, and approximately half the number compared with those cavitating on day 5. The resulting blastocysts seemed to be of equivalent quality when vitrified and transferred at a future date because the pregnancy rates were similar between the two groups. The authors quite reasonably conclude that fresh transfer of delayed morulae is the prudent alternative to continued culture and vitrification, a valuable piece of information derived from good numbers. Is there a larger lesson to be gained? Likely so, because it is nearly a universal strategy in embryo culture to give a sub-par embryo all the time that is available to become what we think is acceptable. That pregnancies are obtained from the transfer of embryos cryopreserved on day 7 of culture attests to it being a seemingly reasonable approach. Perhaps it is not so in all cases, however, and the timing of cavitation may indeed be one marker that portends embryonic fate upon continued culture vs. stopping short with fresh transfer. This places embryologists in a similar professional situation as musicians, trapeze artists, and demolition experts, whereby decisions regarding timing are everything. Embryos that gently apply the brakes may be very different entities from those that do so abruptly, and our laboratory and clinical decisions should be informed by them rather than assuming that additional time in culture yields additional quality. Developmental potential of slow-developing embryos: day-5 morulae compared with day-5 cavitating morulaeFertility and SterilityVol. 111Issue 1PreviewTo describe and compare the ongoing pregnancy rate between morulae and cavitating morulae (CAVM) transferred on day 5, to describe and compare the blastulation rate between day 5 morulae and CAVM, and to describe the pregnancy rate of these slow-developing blastocysts during a frozen embryo transfer (FET) cycle. Full-Text PDF

When considering the multitude of elements that influence success in human assisted reproductive technology (ART), there is something upon which we all can agree: it is complicated. Specifically, if we consider in total the influential items for the laboratory, the clinical environment, ovarian stimulation and ovulation triggering, the retrieval room, embryo transfer technology and patient-specific characteristics, it is easy to tabulate over 200 factors that can alter outcome (Pool et al, 2012Pool T.B. Schoolfield J. Han D. Human embryo culture medium comparisons.in: Smith G.D. Swain J.E. Pool T.B. Embryo Culture, Methods and Protocols. Methods in Molecular Biology 912. Humana Press, New York2012: 367-386Crossref Scopus (25) Google Scholar). It gets no easier if the analysis is restricted to the laboratory or even to a consideration within the laboratory of the role of environmental factors such as air quality. It is this complexity – the impossibility of isolating a single factor for experimental evaluation while holding all others constant – that eliminates the randomized controlled trial as the appropriate tool for defining air quality in the IVF laboratory (Cohen, Alikani, 2013Cohen J. Alikani M. Evidence-based medicine and its application in clinical preimplantation embryology. Reprod.Biomed. Online. 2013; 27: 547-561Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). This very much relates to the significance of the ‘context’ of a given intervention rather than the intervention per se – a well-known topic of intense discussion regarding the limitation of evidence-based medicine research strategy (Fauser, 2016Fauser B.C.J.M. What makes RBM online special: the chief editor point of view.Reprod. Biomed. Online. 2016; 33: 535-536Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). The difficulty in obtaining direct evidence, along with the unethical aspect of experimentally exposing human gametes and embryos to potential environmental toxins, were two of the premises for a meeting of 13 experts representing academia, private practice and commercial enterprises convened to identify and discuss laboratory environmental influences. The results of their deliberations are presented in this issue of RBM Online in the article ‘Cairo consensus on the IVF laboratory environment and air quality: report of an expert meeting’ (Mortimer et al, 2018Mortimer D. Cohen J. Mortimer S.T. Fawzy M. McCulloh D.H. Morbeck D.E. Pollet-Villard X. Mansour R.T. Brison D.R. Doshi A. Harper J.C. Swain J.E. Gilligan A.V. Cairo consensus on the IVF laboratory environment and air quality: a report of an expert meeting.Reprod. Biomed. Online. 2018; https://doi.org/10.1016/j.rbmo.2018.02.005Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) in which the authors list 50 consensus points for the provision of safe air quality, covering laboratory design, construction, operation and engineering controls. Concerns about the adverse effects of poor air quality upon human embryogenesis have been voiced routinely in the literature since the 1990s, some of which are amalgamated in two timely reviews (Morbeck, 2015Morbeck D.E. Air quality in the assisted reproduction laboratory: a mini-review.J. Assist. Reprod. Genet. 2015; 32: 1019-1024Crossref PubMed Scopus (19) Google Scholar, Thomas, 2012Thomas T. Culture systems: air quality.in: Smith G.D. Swain J.E. Pool T.B. Embryo Culture, Methods and Protocols. Methods in Molecular Biology 912. Humana Press, New York2012: 313-324Crossref Scopus (4) Google Scholar). In addition to providing a historical context, Thomas, 2012Thomas T. Culture systems: air quality.in: Smith G.D. Swain J.E. Pool T.B. Embryo Culture, Methods and Protocols. Methods in Molecular Biology 912. Humana Press, New York2012: 313-324Crossref Scopus (4) Google Scholar describes quantitative analytical tests for airborne contaminates including US Environmental Protection Agency (EPA) method EPA TO15, capable of identifying 97 of the 187 hazardous airborne pollutants listed by the EPA, and EPA TO11 which focuses on identifying ketones and aldehydes, including formaldehyde. He further suggests design controls for the laboratory to control airborne contamination and to improve other operational practices relating to aspects such as staging areas, laboratory access and cleaning agents. More recently, Morbeck, 2015Morbeck D.E. Air quality in the assisted reproduction laboratory: a mini-review.J. Assist. Reprod. Genet. 2015; 32: 1019-1024Crossref PubMed Scopus (19) Google Scholar examined eight studies concerning the effects of air filtration, both particulate and chemical, upon clinical outcome in IVF. Endpoints ranged from the evaluation of clinical pregnancy rate alone (Jindal et al, 2008Jindal S.K. Polotsky A.J. Buyuk E.R. Lieman H.J. Gilligan A. Improved pregnancy rates following introduction of engineering controls of lab air quality.Fertil. Steril. 2008; 90: S403Abstract Full Text Full Text PDF Google Scholar, Knaggs et al, 2007Knaggs P. Birch D. Drury S. Morgan M. Kumari S. Sriskandakumar R. Avery S. Full compliance with the EU directive air quality standards does not compromise IVF outcome.Hum. Reprod. 2007; 22: i164-i165Google Scholar) to the inclusion of fertilization, cleavage and blastocyst rates plus clinical pregnancy, implantation and live birth rates (Munch et al, 2015Munch E.K. Sparks A.E. Duran H.E. Van Voorhis B.J. Lack of carbon air filtration impacts early embryo development.J. Assist. Reprod. Genet. 2015; 32: 1009-1017Crossref PubMed Scopus (14) Google Scholar). As Morbeck indicates, while each study noted an improvement in either laboratory and/or clinical outcomes following filtration, nearly all studies housed comparisons that were unmatched and retrospective, thus leaving the possibility that the results were related to another uncontrolled variable and therefore circumstantial. The report by Munch et al. is of particular interest as it includes laboratory and clinical data from a period when carbon filtration of laboratory air was present, an interim period when it was inadvertently absent, and then again when carbon filtration was restored. A consideration of all cycles showed that the restoration of carbon filtration resulted in a return to the fertilization, cleavage and blastocyst rates achieved in the original carbon-filtered period from a depressed rate seen during the period when there was a lack of filtration. While this was also true when only ICSI cycles were considered, it did not hold for conventional insemination cycles. In those, neither fertilization nor blastocyst conversion rates were affected by the absence of carbon filtration. Despite the effects seen upon laboratory parameters, there was no significant effect of a lack of carbon filtration upon clinical pregnancy rate, implantation rate and live birth rate when all three periods were compared. In a different setting, Esteves, Bento, 2013Esteves S.C. Bento F.C. Implementation of air quality control in reproductive laboratories in full compliance with the Brazilian Cells and Germinative Tissue Directive.Reprod. Biomed. Online. 2013; 26: 9-21Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar reported live birth rates increased while miscarriage rates decreased, both significantly, when IVF patients were treated in a new facility constructed in compliance with cleanroom standards for particulates and volatile organic compounds (VOC) as dictated by the Brazilian Cells and Germinative Tissue Directive. As in earlier studies, the retrospective comparison of these 2060 patients was made with a group of 255 patients treated earlier by the same practice in a conventional facility. To reduce the number of variables influencing outcome, Heitmann et al, 2015Heitmann R.J. Hill M.J. James A.N. Schimmel T. Segars J.H. Csokmay J.M. Cohen J. Payson M.D. Live births achieved via IVF are increased by improvements in air quality and laboratory environment.Reprod. Biomed. Online. 2015; 31: 364-371Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar compared cycles performed in a new facility, where strategic engineering designs were employed, with those carried out in an old facility housed in operating room space where air was being supplied by the operating room air handler without consistent positive pressure being attained in the laboratory. Environmental improvements were extensive in the new facility and included a dedicated air filtration system employing humidity control, paper filtration, UV light exposure and filtration through a mixed bed of activated charcoal and potassium permanganate prior to HEPA (high efficiency particulate air) final filtration. Construction allowed for a sealed environment, low to no VOC-emitting materials, positive pressure and limited access by authorized personnel. Further, laboratory equipment utilized was the same in both facilities, as were the physicians, embryologists, nurses and protocols. The rate of both embryo implantation and live birth improved significantly in the new facility. Despite the retrospective nature of the comparison, it is likely that this is as close to a valid comparison of environmental influences in the IVF laboratory as can be made. To an experimentalist, a consensus of opinions carries dubious weight given that it is not a component of the scientific method. Perhaps Bertrand Russell expressed it best in this partial quotation from his 1929 treatise, Marriage and Morals, when he quipped ‘The fact that an opinion has been widely held is no evidence whatever that it is not utterly absurd…’. But in the Cairo consensus we have specific suggestions from a panel of scientists deeply experienced in laboratory design, construction, operation and air quality measures with the safety of human gametes and embryos as the sole endpoint. Anyone considering new laboratory construction, re-vamping of an older facility or seeking appropriate ways to ensure a high-quality environment can now find a practical compendium of what to consider and what to do within a single document. Technology is not free and we owe it to patients to maximize accessibility to treatment as best we can. Adding costs to the construction and operation of our facilities, when unwarranted, thwarts that goal. A responsible part of providing a safe environment is the inclusion of economic considerations in our planning and addressing whatever the environmental testing suggests is required; however, whatever remedy is needed is undoubtedly covered by the Cairo consensus.

Sperm DNA fragmentation (SDF) represents a perplexing biological phenomenon that can, at an elevated level, interfere with either initiating and/or maintaining pregnancy. Although we are in our third decade of studying this problem, few investigators have attempted to assess the value of the various available assays for SDF with the specific aim of amalgamating them into practice guidelines for managing SDF. Agarwal, in collaboration with urological experts from North and South America, has done just that, employing a number of clinical scenarios involving SDF to produce some evidence-based, practical guidelines for patient management (1). But if in our third decade of investigation, why are management guidelines so difficult to come by for patients with elevated SDF? First, it should be emphasized that SDF is an exceedingly complex process involving not one, but a number of causative mechanisms that can generate a variety of insults to the integrity of sperm DNA. Sakkas and Alvarez (2) have described six main mechanisms that can damage both sperm nuclear as well as mitochondrial DNA. Included in this list are testicular apoptotic processes during spermatogenesis, aberrations during the events of chromatin re-modeling, induction by exogenous caspases and nucleases plus damage by chemo- and radiotherapy as well as environmental toxicants. Perhaps the most robust events occur in the epididymis in response to insult by reactive oxygen species. That epididymal and ejaculated sperm have higher amounts of DNA fragmentation than testicular sperm is now well documented (3-5). What is not known are the testicular events that render sperm labile to chemical radicals traversing the epididymis. Secondly, the insults can produce single-stranded breaks, double-stranded breaks and/or nucleotide damage. Not all are detected by the same tests, the different types of damage are repaired to different degrees by processes resident in the oocyte and thus, do not have the same prognosis. Likely though, it is the tests used to detect and measure SDF, along with their varying levels of complexity, that introduce the bulk of the conflicting data and conclusions common in the SDF literature.

Sir, Ensuring and monitoring the health of children conceived through IVF is paramount, of that there can be no compromise. Hence, we found it perplexing to read the paper by Kleijkers et al. (2016) on the effects of culture media and birthweight. This is not the first randomized controlled trial (RCT), being predated by others, including the prospective trial by Carrasco et al. (2013) who reported the same weight for G5 (3213 g) and Cook (3154 g) media, but did not identify either as resulting in low birthweight. Similarly, the Kleijkers paper does not mention studies such as that of De Vos et al. (2015) whose data on birthweight are in line with those of Carrasco et al., nor the data of many the retrospective studies on birthweights, including Eskild et al. (2013) who reported a mean birthweight of 3444 g following culture in G5 medium. A recent study by Maas et al. (2016) including 6265 singleton live births showed no impact of media over an 18-year period. In fact, they concluded that the fresh versus frozen birthweights seemed was the only significant factor impacting IVF singleton birthweight. Of significance, this study was not powered for any conclusions about birthweight; the primary endpoint was live birth rate. Further, the birthweights of twins in both media in the Kleijkers study are the same, questioning how one medium can differentially affect the growth trajectory of singletons. It is therefore curious to speculate about why the birthweight difference of 158 g, restricted to singletons, was selected for comment when most outcomes favoured G5. It is apparent from Table 4 that the foetal growth trajectories of the two groups were not different, which suggests that the difference in birthweight is related to the higher rate of births <37 weeks in the G5 group. The isolated effect on gestationally mediated reduction in birthweight for singletons is consistent with residual confounding by medical interventions, e.g. induction of labour. The analysis is less than ideal in other regard as they adjusted for twinship, but not for clustering within site, which are clearly very different. They could also have adjusted for source and concentration of albumin, day of transfer, number of embryos transferred and cryopreservation, but did not. This would require more power, but the randomization is not presented by site, so it is not possible from the presented data to know if randomization was effective for potential confounders. The study was therefore underpowered for the task. Of further concern to us was not only the large number of variables in this trial, but the use of the medium HTF, a medium that became obsolete at the turn of the century due to its lack of amino acids, and which in our view, has no future place in human ART. From our perspective, the use of this medium in a trial is completely unacceptable, and explains why only 15% of embryos, from patients with an average age of 37, implanted in this group. What also becomes apparent from the data set is that significantly more patients required a second and third ovarian hyperstimulation in the HTF group (Supplementary data, Table 1); a patient-related risk that is completely ignored. Finally, the statement that hyaluronan was a new inclusion of the G media is erroneous given that it has been in all G series media since 2000. The companion paper by Sunde et al. (2016) subsequently argues that the differences in culture media formulations evoke the ‘Barker hypothesis’, now better known as the ‘Developmental Origins of Health and Disease (DOHaD)’, which proposes that birthweight (more accurately leanness at birth for gestational age), is an indicator of subsequent adult metabolic health. Initially controversial when proposed, the hypothesis has since received worldwide confirmation and acceptance. This includes many epidemiological studies in humans and experimental animal studies that demonstrate the peri-conception period is a sensitive time, where mechanisms, most likely epigenetic in nature, operate that determine fetal growth. However, we argue that the Kleijkers paper does not prove that differences in birthweight are induced by differences in media formulation, primarily because the Kleijker et al. paper is so highly confounded as an RCT, hence the relatively small difference in birthweight could readily be due to the myriad of confounding factors. Centrally against the DOHaD argument, the incidence of small for gestational age (SGA) infants did not differ between the groups. There are important independent long-term developmental effects of birthweight, SGA and prematurity. Unfortunately, these factors are conflated in the reported RCT. We agree with Sunde et al. (2016) that current media manufacturers should be required to inform all end-users of formulation changes. We also agree with Sunde et al.’s position that more appropriately conducted RCTs are required. We argue that a debate on disclosure of formulation should be held with media manufacturers, so their point of view is represented and, therefore, the debate is balanced. However, raising concerns about how differences in formulations are linked to long-term adult health outcomes on the basis of this one insufficient RCT is premature.

Purpose The objective of this study was to offer a new treatment approach for sperm retrieval simultaneously with tumor resection in azoospermic men with congenital adrenal hyperplasia (CAH), orchialgia, and bilateral testicular adrenal rest tumors (TARTs) who fail to respond to medical treatment. Methods This is a retrospective chart review from a couple’s fertility center. Results Between May 2013 and May 2015, two azoospermic men with CAH and bilateral TARTs, with orchialgia, and desire to conceive underwent bilateral TART resection in the same surgical setting as sperm retrieval after remaining azoospermic with normalization of gonadotropins with treatment with human chorionic gonadotropin (hCG). Both men had adequate sperm retrieved for in vitro fertilization/intracytoplasmic sperm retrieval (IVF/ICSI) at the time of bilateral TART resections. They had complete TART resections with resolution of orchialgia. The wife of one patient had a successful pregnancy with use of retrieved sperm resulting in a live birth, and the sperm from the other man is cryopreserved for future use. Conclusions It is feasible to perform successful sperm retrieval simultaneously with TART resection in azoospermic men with CAH after medical treatments with persistent azoospermia, rather than subjecting these men to multiple invasive procedures.

The purpose of the study was to report a case of live birth following donor oocyte in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) in which the oocyte donor herself was conceived via IVF. To our knowledge, such a case has not been previously reported. Retrospective chart review; this case is reported after chart review of a successful outcome. A 42 year-old woman, with diminished ovarian reserve, and her husband desired to conceive. She underwent a fresh IVF/ICSI cycle with her own oocytes, which unfortunately was not fruitful in terms of pregnancy or cryopreserved embryos. The couple was counseled regarding the option of donor oocytes, and they elected to proceed with a fresh cycle of donor oocyte IVF/ICSI. The couple selected an anonymous oocyte donor from a donor agency who was a first-time oocyte donor and, interestingly, was conceived via IVF herself. The fresh donor oocyte/IVF/ICSI cycle did not result in pregnancy; however, two supernumerary blastocysts were cryopreserved for future cycles. The recipient’s subsequent frozen-thawed embryo transfer (FET) resulted in a singleton gestation and live birth. An oocyte donor who was conceived via IVF had good ovarian response to stimulation, a good number of oocytes retrieved, and the formation and cryopreservation of blastocysts which, in a subsequent FET cycle, resulted in pregnancy and live birth for a recipient couple. To our knowledge, this is the first case reported of live birth with the use of donor oocytes from an oocyte donor who herself was conceived via IVF.

Objective: To determine whether antimullerian hormone (AMH) levels predict the availability of good-quality supernumerary blastocysts for cryopreservation.Design: Retrospective study.Setting: Two fertility centers.Patient(s): First fresh IVF cycles (n = 247) grouped as follows: 40 women <35 year old with AMH <1 ng/mL and 77 women with AMH 1-4 ng/mL; 62 women >= 35 year old with AMH <1 ng/mL, and 68 women with AMH 1-4 ng/mL.Intervention(s): AMH level measured before IVF with ovarian stimulation protocols based on patient age and AMH level, including short gonadotropin-releasing hormone (GnRH) agonist, GnRH antagonist, or GnRH agonist microdose flare; supernumerary good-quality blastocysts cryopreserved on days 5 or 6 after retrieval.Main Outcome Measures(s): Supernumerary good-quality blastocysts for cryopreservation in relation to AMH levels.Result(s): Among women <35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocysts for cryopreservation between the groups with AMH <1 ng/mL and AMH 1-4 ng/mL (30.0% vs. 58.4%) when adjusted for age. Among women >= 35 years of age, there was a statistically significant difference in the number of patients with supernumerary good-quality blastocyst cryopreservation between groups with AMH <1 ng/mL and AMH 1-4 ng/mL (16.1% vs. 42.6%), when adjusted for age.Conclusion(s): Low AMH levels are associated with a statistically significantly lower likelihood of blastocysts for cryopreservation as compared with higher AMH levels. This effect was seen among women both <35 and >= 35 years of age. Patient counseling should include realistic expectations for the probability of good-quality supernumerary blastocysts available for cryopreservation. (C) The Authors. Published by Elsevier Inc. on behalf of the American Society for Reproductive Medicine.

Until 1999, men with non-obstructive azoospermia (NOA) did not have an option to conceive using their sperm [1]. Since the advent of microdissection testicular sperm extraction (MicroTESE), men with NOA have a chance to conceive by using their sperm for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). With more widespread use of serum antimullerian hormone (AMH) testing to assist in the evaluation of diminished ovarian reserve (DOR), the controversy of appropriate options for couples with both of these obstacles has arisen.

The use of human embryos in research will always provoke controversy and discussion, and rightly so, as it raises complex moral and ethical questions. This was recognised by the government of the UK in 1989 when, whilst drafting legislation to regulate the practice of IVF, it allowed a free vote in Parliament on whether to permit, or to ban outright, embryo research. The legislation that was introduced later in other countries was in some cases permissive, in others less so, and in others prohibitive. Irrespective of the legal position, the views of individuals will vary and frequently conflict: for some absolutists, the ethical difficulties posed are insurmountable, and render any such research immoral; for others, the potential benefits of research using human embryos provide sufficiently powerful moral arguments to outweigh such reservations. The source of research material in this controversial field is currently almost exclusively ‘spare’ or ‘supernumerary’ embryos that arise during therapeutic IVF treatment. For example, of the 13 centres in the UK where research projects were licensed by the Human Fertilisation and Embryology Authority (HFEA) during 2012 and 2013, all but 4 of the 20 projects express the intention to use embryos that have been donated to research, described sometimes as ‘spare’ embryos, that are ‘no longer required by the patients’, and that would ‘otherwise be discarded’ (http://www.hfea.gov.uk/166.html). The situation in the US is somewhat different in that oocytes and human embryos do not fall under the same federal regulations that govern other human materials in clinical laboratories (Pool, 1997Pool T.B. Practices contributing to quality performance in the embryo laboratory and the status of laboratory regulation in the US.Hum. Reprod. 1997; 12: 101-103Crossref PubMed Scopus (6) Google Scholar); in fact, the only federal regulations that apply to human oocytes and embryos are those from the Food and Drug Administration, Department of Health and Human Services, that relate to the eligibility of gamete/embryo donors and recipients from an infectious disease perspective. Further, the ethical acceptability of using human embryos for research has been addressed and confirmed by several national studies and commissions (National Institutes of Health, 1994National Institutes of Health, 1994. Report of the Human Embryo Research Panel: Final Draft, September 27, 1994. Bethesda, MD: National Institutes of Health.Google Scholar). That aside, the vast majority of centres in the US belong to the Society of Assisted Reproductive Technology (SART) and are bound by practices set forth by the American Society for Reproductive Medicine (ASRM). American Society for Reproductive Medicine (Ethics Committee), 2004American Society for Reproductive Medicine (Ethics Committee) Informed consent and the use of gametes and embryos for research.Fertil. Steril. 2004; 82: S251-252PubMed Google Scholar, American Society for Reproductive Medicine (Ethics Committee), 2014American Society for Reproductive Medicine (Ethics Committee) Informed consent and the use of gametes and embryos for research: a committee opinion.Fertil. Steril. 2014; 82: 332-335Google Scholar has indicated (American Society for Reproductive Medicine (Ethics Committee), 2004American Society for Reproductive Medicine (Ethics Committee) Informed consent and the use of gametes and embryos for research.Fertil. Steril. 2004; 82: S251-252PubMed Google Scholar, American Society for Reproductive Medicine (Ethics Committee), 2014American Society for Reproductive Medicine (Ethics Committee) Informed consent and the use of gametes and embryos for research: a committee opinion.Fertil. Steril. 2014; 82: 332-335Google Scholar) that it is the obligation of the clinician to obtain Institutional Review Board approval and informed consent from donors of spermatozoa, oocytes and embryos for research. Simply put, the sourcing of embryos for research from an ongoing therapeutic procedure without approval and consent is unethical in the US. But there are a number of scenarios where embryos are made available for research with proper consent. Such embryos will certainly include many that have been cryopreserved, and that have subsequently been donated to research by patients who have elected not to keep them for use in their own treatment (Hammarberg and Tinney, 2006Hammarberg K. Tinney L. Deciding the fate of supernumerary frozen embryos: a survey of couples’ decisions and the factors influencing their choice.Fertil. Steril. 2006; 86: 86-91Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, Luna et al., 2009Luna M. Boada M. Aran B. Coroleu B. Barri P.N. Veiga A. Couples’ opinions regarding the fate of surplus frozen embryos.Reprod. Biomed. Online. 2009; 19: 11-15Abstract Full Text PDF PubMed Scopus (12) Google Scholar, Melamed et al., 2009Melamed R.M. Bonetti T.C. Braga D.P. Madaschi C. Iaconelli A. Borges E. Deciding the fate of supernumerary frozen embryos: parents’ choices.Hum. Fertil. (Camb). 2009; 12: 185-190Crossref PubMed Scopus (27) Google Scholar). However, until now, the pool of research material has also included ‘fresh’ embryos donated to research from cohorts of embryos generated during patients’ ongoing fertility treatment. That this practice should be re-evaluated is thrown into sharp relief by the work of Poulain et al., 2014Poulain, M., Hesters, L., Sanglier, T., de Bantel, A., Fanchin, R., Frydman, N., Grynberg, M., 2014. Is it acceptable to destroy, or to include in research programmes, human embryos before day 5? Reprod. Biomed. Online 28, 522–529.Google Scholar published in this issue of Reproductive Biomedicine Online, who examined the development of human embryos that, following morphological assessment on day 3 of development, had been designated as ‘poor quality’, and might therefore be judged unlikely to be suitable for use in patients’ treatment. Contrary to their anticipated demise during subsequent culture, a significant proportion (33.6%) of these poor-quality embryos developed into good-quality blastocysts, were cryopreserved, and viable pregnancies have resulted. This important observation is significant both for the routine practice of therapeutic IVF, and for the designation of fresh embryos to the category of ‘otherwise discarded’ and thus as appropriate for allocation to research. Might it be argued that the allocation of fresh embryos to research should be delayed routinely until day 5 or 6 after fertilization, until it has been established beyond doubt that they do not have the potential to form ‘high grade’ blastocysts, and, that all blastocysts that do develop should be cryopreserved for the patients’ use? This practice is already in place in some centres (for example, at the Assisted Conception Unit at Guy’s Hospital in the UK), with the consequence that the only fresh embryos used in research are those diagnosed genetically as ‘unsuitable for transfer’ after preimplantation genetic diagnosis. Throughout the UK and the US, where the practice of ‘extended culture’ and blastocyst-stage embryo transfer is widespread in therapeutic IVF, a significant reduction in the availability of early cleavage-stage human embryos for research is already occurring. Thus, research focusing on these early stages of development will come to rely increasingly on cryopreserved pronucleate and cleavage-stage embryos that have been donated actively by patients who no longer wish to keep them for their own use. The sensitive nature of embryo research requires that those who support it, and those who undertake it, accept that limits must be placed on what is permissible. It is incumbent on those carrying out research using human embryos both to accept and comply with any such limits, and to allay any lingering concerns of the sceptics, be they regarding the extent of any benefits that may result from embryo research, or regarding the provenance of the research material used to achieve them.

Every program of assisted reproduction strives to maximize pregnancy outcomes from in vitro fertilization and selecting an embryo culture medium, or medium pair, consistent with high success rates is key to this process. The common approach is to replace an existing medium with a new one of interest in the overall culture system and then perform enough cycles of IVF to see if a difference is noted both in laboratory measures of embryo quality and in pregnancy. This approach may allow a laboratory to select one medium over another but the outcomes are only relevant to that program, given that there are well over 200 other variables that may influence the results in an IVF cycle. A study design that will allow for a more global application of IVF results, ones due to culture medium composition as the single variable, is suggested. To perform a study of this design, the center must have a patient caseload appropriate to meet study entrance criteria, success rates high enough to reveal a difference if one exists and a strong program of quality assurance and control in both the laboratory and clinic. Sibling oocytes are randomized to two study arms and embryos are evaluated on day 3 for quality grades. Inter and intra-observer variability are evaluated by kappa statistics and statistical power and study size estimates are performed to bring discriminatory capability to the study. Finally, the complications associated with extending such a study to include blastocyst production on day 5 or 6 are enumerated.

The fi rst tissue culture media were developed nearly 150 years ago by Ludwig and Ringer. These were simple salt solutions, which were initially based on the chemical properties of blood serum. The second generation of culture media was developed more than a century later, in the 1970s, aiming to mimic the reproductive environment. In the 1990s, simplex optimization was used to design the third group of media, to some extent ignoring existing formulations and principles. Simultaneous with the development of culture media, it became evident that it was necessary to carefully control the culture conditions, including temperature, pH, osmolarity, and air quality. Equally important was the development of instruments and tools speci fi cally designed for cell tissue culture such as the inverted microscope, the incubator, the Petri dish, sterile plasticware, the laminar fl ow cabinet, and air fi ltration equipment.