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Cemeare.ptJ Assist Reprod GenetDOI 10.1007/s10815-012-9793-z ASSISTED REPRODUCTION TECHNOLOGIES Influence of post-thaw culture on the developmentalpotential of human frozen embryos Mafalda L. Rato & António Gouveia-Oliveira &Carlos E. Plancha Received: 4 February 2012 / Accepted: 30 April 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com This comparison was corrected for the most common Purpose Apart from freezing/thawing related cryodamage, confounding factors such as maternal age at oocyte pick- several additional factors have been identified as major up, number of transferred embryos, developmental day at players in the reduction of success rates after frozen embryo freezing, blastomere survival after thawing, catheter used transfers. The post-thaw culture is particularly relevant as it for transfer and year of procedure.
may amplify environmental influences over a stressed em- Results Implantation and live birth rate per embryo trans- bryo. In the present study the influence of the post-thaw ferred were inversely related to the duration of the post-thaw culture duration on the implantation and developmental culture, as diminishing this period significantly increased potential of cleavage stage embryos was evaluated.
both rates. Moreover, no advantage could be found for a Methods In this retrospective evaluation, that spanned an long post-thaw culture period, even for embryos with ob- 8-year period, 631 frozen-thawed embryos were allocated to served mitotic activity.
one of two study groups, depending on their post-thaw Conclusion This retrospective analysis indicates that a short culture period: 1) the long (18–24 h), or 2) the short post-thaw culture period is associated with higher implanta- (2–5 h) culture group. Groups were compared regarding tion and live birth rates per embryo. This study supports implantation rate and live birth rate per embryo transferred.
selection of frozen-thawed embryos strictly based on blasto-mere cryosurvival and raises the hypothesis that environmen-tal factors may have an important role on embryo implantation Capsule A short post-thaw culture period allows expression of higher and developmental potential during post-thaw culture.
developmental competence (higher implantation and live birth rates perembryo transferred), in human cleavage stage embryos.
Keywords Embryo cryopreservation . Blastomere M. L. Rato : C. E. Plancha (*) proliferation . Culture environment . Implantation rate .
Centro Médico de Assistência à Reprodução—CEMEARE,Rua Alfredo Mesquita, 2E, Developmental competence 1600-922 Lisbon, Portugale-mail: [email protected] A. Gouveia-Oliveira Departamento de Bioestatística, Faculdade de Ciências Médicas,Universidade Nova de Lisboa, Currently, the two most common procedures to select em- Campo dos Mártires da Pátria 130, bryos for frozen embryo transfer (FET) differ in the duration 1169-056 Lisbon, Portugal of the post-thaw culture: one relies upon the observation of blastomere proliferation, requiring a longer culture, gener- Unidade de Biologia da Reprodução, ally overnight; the other relies upon the observation of Instituto de Histologia e Biologia do Desenvolvimento, blastomere survival after thawing, requiring a shorter cul- Av. Professor Egas Moniz,1649-028 Lisbon, Portugal ture. The effectiveness of one strategy over another is still J Assist Reprod Genet not clear as few studies have compared large series of from the cryopreservation of supernumerary embryos gen- embryos over a long time period. Additionally, all published erated in the course of IVF/ICSI cycles. Irrespective of the results are influenced by biasing factors relating to the number of FET cycles derived from each ovarian pick-up population demographics and to events described as modu- (OPU), only embryos derived from the first FET were eli- lators of FET outcome [, , , gible for the study. Only viable embryos were considered— Although FET is generally considered a well established defined as having retained ≥50 % of their initial number of technique, a large number of studies concentrate on the blastomeres intact upon thawing.
reasons for its lower success rate when compared to fresh Concerning the time span of the study, two main periods embryo transfers Major contributors to such diver- reflecting two different post-thaw methodologies were tra- gence have been extensively discussed and, as an attempt to versed. The first period extended mainly from May 2001 to elevate frozen embryo transfer (FET) outcome, the post- June 2005 and coincided with the established tendency to thaw assessment of mitotic resumption became a widely perform an overnight post-thaw culture and select embryos employed strategy for embryo grading and selection [, for transfer based on their mitotic activity. The second , ]. As a consequence, the specific effect of period extended mainly from July 2005 to December 2008 post-thaw culture duration has remained neglected by most and significantly represented our new tendency to reduce FET studies. In our centre, until 2005, post-thaw embryo the culture period between thaw and transfer, relying only selection was preferentially based on mitotic resumption.
on post-thaw survival for embryo selection.
However, FETs were most frequently non-elective and em-bryos surviving the thawing process were generally the same IVF/ICSI procedures embryos selected for transfer the following day, irrespective ofsubsequent mitotic resumption. Interestingly, after an internal Patients undergoing IVF/ICSI cycles were subjected to con- evaluation of our FET program, a tendency towards a better trolled ovarian stimulation using a GnRH agonist (Decapetyl, outcome following transfer of embryos thawed on the same Ipsen Pharma Biotech, Signes, France) or antagonist (Orgalu- day as transfer was identified. In light of such observation, tran, Organon/Schering-Plough, UK; or Cetrotide, Serono, thawing began to preferentially be performed on the same day London, UK) for hypolalamic down-regulation; human men- as transfer and embryo post-thaw selection based on mitotic opausal (Menopur, Ferring GmbH, Kiel, Germany) or recom- activity was no longer a priority.
binant FSH (Gonal-F, Serono, UK; or Puregon, Organon/ The current understanding that available embryo culture Schering-Plough, UK) for ovarian stimulation; and hCG systems, although aiming to mimic the physiological environ- (Pregnyl, Organon/Schering-Plough, Oss, Holand; or ment, do not replace it completely , supported the modifi- Ovitrelle, Serono, London, UK) for induction of oocyte mat- cation of our post-thaw culture protocol. It additionally raised uration. After ovarian puncture COCs were incubated in IVF the hypothesis that frozen-thawed embryos may be particular- medium (all culture media from MediCult/Origio, Jyllinge, ly sensible to sub-optimal environmental influences during Denmark). Zygotes and day 2 embryos were cultured in ISM1 post-thaw culture, possibly interfering with potential to im- medium and transferred to ISM2 medium at day 3. Cleaving plant and development to a live birth. In order to explore the embryos were scored every day for blastomere number, cleav- specific effect of post-thaw culture duration upon implantation age plane and degree of fragmentation and graded according- and live birth rates, we conducted a retrospective evaluation ly. Higher grade embryos were selected for fresh embryo spanning an 8-year period. Specifically, we compared transfer. Surplus embryos presenting an adequate relation the influence of two distinct post-thaw culture lengths, between blastomere number and developmental day, a correct 2–5 h versus 18–24 h, on embryo implantation and full cleavage plan and a maximum of 20 % fragmentation were term developmental potential using a statistical approach designed to control for the influence of common biasingfactors.
Cleavage stage embryo slow freezing and rapid thawing The freezing-thawing procedures and media remained un- Materials and methods changed during the complete study period. According to theslow freezing protocol, embryos were sequentially equilibrat- Embryo inclusion and study period ed at room temperature in a HEPES-buffered medium con-taining cryoprotectants up to a final concentration of 1.5 M This retrospective study evaluated all frozen-thawed embry- 1,2-propanediol (PROH) and 0.1 M sucrose (Medicult/Origio os derived from FET cycles initiated at Centro Médico de embryo freezing pack). Embryos were loaded into ministraws Assistência à Reprodução (CEMEARE) in Lisbon, Portugal, (CryoBioSystems, France) and cooled using a programmable during an 8-year period. FETs included in this study resulted freezer (MiniCool, AirLiquid, France). The cooling program
J Assist Reprod Genet started at 20°C and ran as follows: temperature was taken to Before FET, embryos were rechecked for viability, placed −7°C at a rate of 2°C/min and manual seeding was performed in pre-equilibrated Universal Transfer Medium (UTM) and by touching the extremes of the straw with a nitrogen-cooled loaded into an appropriate catheter (Frydman or TDT, Labo- forceps. From that point on, the temperature was slowly ratoire CCD, Paris; or Sydney IVF embryo transfer set, Cook decreased at 0.3°C/min to −30°C. A final and very fast drop OB/GYN, USA). Regarding mitotic activity, three types of to −150°C was accomplished decreasing the temperature at a FETs occurred: FETs in which all embryos transferred had rate of 50°C/min. Straws were then plunged into liquid nitro- cleaved during the culture period (with observed mitosis); gen and stored until thawing.
FETs in which none of the embryos transferred had cleaved Rapid thawing was achieved by quickly placing the straws during the culture period (without observed mitosis); and on hand for a minimum of 30 s. Embryos were then dis- FETs in which some of the embryos had and some had not charged to a 4-well dish and washed from the cryoprotectants cleaved during the culture period. In this last group of FETs, at room temperature, using sequential thawing media embryos could not be assigned to either group and were (Medicult/Origio embryo thawing pack). According to their considered non-informative regarding mitotic resumption. In developmental day upon freezing, embryos were placed in the short culture group, mitotic activity was not assessed due appropriate medium, checked for blastomere survival under to biological inadequacy of such observation (Fig. an inverted microscope and incubated until transfer. Long Clinical pregnancy was defined by the presence of a post-thaw incubation consisted of an overnight culture for an gestational sac with a fetal heartbeat on ultrasound exami- 18–24 h period before transfer. Short post-thaw incubation nation at 6 weeks of gestation.
consisted of a culture for a 2–5 h period before transfer.
Statistical analysis Uterine transfer of frozen-thawed embryos The primary efficacy endpoints were the implantation rate Embryo replacement to the uterus was performed following (IR—the number of gestational sacs per number of embryos endometrium preparation using Leucoprolide (Decapetyl transferred) and the live birth rate per embryo (LBR/E—the depot 0,1 mg/ml, Ipsen Pharma Biotech, Signes, France)— number of newborns per number of embryos transferred).
a GnRH agonist, for hypothalamic suppression and 17β- Secondary efficacy endpoints were pregnancy, multiple preg- estradiol (Estrophem, Novo NordisK, Novo Allé, Denmark) nancy, gestational sac involution and delivery rates. In addi- in order to mimic the natural occurring estrogens until the tion, we looked into the relevance of observing mitotic activity endometrium reached 8 mm thick and the serum estradiol to implantation rate and the live birth rate per embryo.
levels reached ≥150 mg/dl. From this stage on, progesterone Differences between groups were tested with multiple (Utrogestan, Jaba Recordati, Sintra, Portugal) was added to linear regression for interval-measured endpoints and logis- the previous therapeutic scheme for endometrium support tic regression for binary endpoints. Whenever endpoints and embryo transfer was scheduled referred to episodes, analyses were clustered by subject Fig. 1 Flow chart summarizingthawed embryo accountabilityof the study. Implantation ratesand live birth rates per embryowere compared between thelong and the short culturegroups. These outcomes wereadditionally compared betweenembryos with observed mitoticresumption from the longculture group and embryoswithout observed mitosis bothin the long and short culturegroups
J Assist Reprod Genet Table 1 Demographic, clinical and embryological characteristics of FETs in the two different post-thaw culture groups Short culture group Long culture group Maternal age at OPU, mean (SD) Developmental day at freezing, n (%) Post-thaw surviving embryos, % No. Transferred embryos, mean (SD) Catheter used for transfer, n (%) Year of procedure, n (%) and robust "Sandwich" (Huber-White) standard errors were than in the long culture group (IR: 11.5 % vs 4.0 %; LBR/E: used to account for non-independence within subjects. In 9.7 % vs 1.7 %) (Fig. The adjusted odds-ratio for im- order to control for systematic differences between groups, plantation rate in the short to the long culture group was all analyses were adjusted for the following potential con- 2.32 (95 % confidence interval 1.04 to 5.17, p00.04) and for founding factors: maternal age at OPU, number of embryos live birth rate was 2.96 (95 % confidence interval 1.08 to transferred, day of embryonic development at freezing, blas- 8.15, p00.04) (Table ).
tomere cryosurvival after thawing, catheter used for transfer Regarding the secondary efficacy variables (Table and and procedure year. All tests are two-tailed. Results are Fig. ), although the observed differences were in favor of reported as adjusted odds-ratios (AOR) with 95 % confidence the short culture group in the pregnancy rate (21.4 % vs intervals (95 % CI). Differences were considered significant if 12.5 %, AOR 2.32, 95 % CI 0.89 to 6.02), they did not p<0.05. Statistical analysis was performed using STATA 11 reach statistical significance (p00.085). Differences were (Stata Corporation, College Station, Texas). Whenever appro- also not statistically different either in the rate of multiple priate, writing and analysis followed the STROBE pregnancies (p00.085), neither in the gestational sac involu-tion rate (p00.86).
Between May 2001 and December 2008, 631 viable embryosout of a total of 1046 were analyzed, 176 in the long culturegroup (18–24 h of post-thaw culture) and 455 in the shortculture group (2–5 h of post-thaw culture). Particularly, theembryos analyzed derived from the first FET of 265 OPUs.
Considering the culture groups, 70 FETs were included in thelong and 195 in the short culture group. As expected in aretrospective study, there were differences between the studygroups regarding demographic, clinical and embryologicalcharacteristics of the populations (Table In order to accountfor their influence on the results, all analyses were adjusted formean maternal age at OPU, number of transferred embryos,developmental day at freezing, catheter used for transfer andyear of procedure.
Fig. 2 Influence of distinct post-thaw culture periods. Embryos cul-tured for a long (18–24 h) or a short (2–5 h) period exhibit very distinct Both implantation rate and live birth rate per embryo outcomes: the unadjusted probability of frozen embryos to implant and transferred were found to be significantly higher in the short develop to term significantly increases if briefly cultured J Assist Reprod Genet Table 2 Outcome of FETs following distinct post-thaw culture periods Table 3 Relevance of observing mitotic activity. Comparison of the in the short to the long culture group outcomes of embryos transferred after observed mitotic resumption inthe long culture group (n061) to embryos without mitotic activity in the short and in the long culture groups Implantation rate Live birth rate per embryo Short culture (n0455) Implantation rate Multiple pregnancy rate Live birth rate per embryo Gestational sac involution Long culture (n029) Implantation rate Live birth rate per embryo In order to assess the relationship between blastomere Cleavage stage embryos were allocated to different transfer groups mitotic activity and embryo developmental potential, we according to mitotic resumption: long culture group, where mitotic compared the outcome of embryos transferred after ob- resumption was observed in all embryos transferred; long culture served mitotic resumption in the long culture group (61 group, where no mitotic resumption was observed in all embryostransferred; short culture group, where no mitotic resumption was embryos) and embryos transferred after a short post-thaw expected to occur during the culture period culture (in which no mitotic activity was observed, 455embryos). These groups were not statistically different in the mean number of embryos transferred (2.2±0.7 and 2.3±0.7; p00.43) and mean maternal age at OPU (32.0±4.6 and The results of this retrospective study indicate that a short post- 33.5±4.4; p00.06). No statistical advantage was found from thaw culture period is associated with higher implantation and transferring embryos cultured for a long post-thaw period and live birth rate per embryo. To our knowledge, this is the first with proven mitotic division, over embryos cultured for a study to identify a long post-thaw culture period as being reduced length of time regarding implantation rate (odds ratio responsible for loss of embryo implantation and developmental 1.99, p00.20) and live birth rate per embryo (odds ratio 1.97, potential. Additionally, this study reinforces the advantages of p00.26). Additionally, using only data from the long culture a frozen-thawed embryo selection approach strictly based on group, we also compared the developmental potential of em- blastomere cryosurvival , ].
bryos with proven (n061) or not observed (n029) blastomeremitotic activity, since there was a similar mean number ofembryos transferred (2.4±0.9, p00.43) and a mean maternal The observed differences between study groups rely age at OPU (33.9±3.9, p00.20). Again, no statistically sig- on a strong statistical plan and are independent nificant difference was found in implantation rate (odds ratio of common biasing factors 1.88, p00.71). The numbers were however insufficient tocompare the live birth rate per embryo (Table ).
Although the conclusions from this study are based on retro-spective data and have the inherent limitations of retrospectivestudies, there are strengths in its design that afford strong Implantation rate confidence on the results. First, the study was based on a large Live birth rate per embryo sample of transferred embryos spanning a period of 8 years.
Second, the quality of data is high, without missing data or a Multiple pregnancy rate single case lost to follow up. Third, to overcome the lack ofrandomization between groups, the analysis accounted for a Gestational sac involution rate number of possible confounding factors of FET outcomes such as maternal age at OPU, number of embryos transferred,day of embryonic development at freezing, blastomere cryo-survival after thawing, catheter used for transfer and year of procedure. The latter two variables are often neglected but Fig. 3 Adjusted odds-ratios and 95 % confidence intervals of the importantly reflect increasing operator (embryologist and cli- study variables in the short to the long culture group. Six potential nician) experience and the continuous improvement of equip- confounding factors were controlled: maternal age at oocyte pick-up, ments and devices, such as embryo transfer catheters.
number of embryos transferred, day of embryonic development at Particularly, adjusting the analysis for the year of procedure freezing, blastomere cryosurvival after thawing, catheter used fortransfer and year of procedure was essential as the superiority of one culture duration (long or J Assist Reprod Genet short), could be biased by the preferential association with a present study, we suggest that "post-thaw culture duration" time period (from 2001 to 2004 and from 2005 to 2008). In should be considered as another confounding factor, strongly addition, the analysis accounted for the clustering of episodes influencing developmental potential and consequently FET (transfers) within subjects (couples) and adjusted standard errors for possible non-independence of episodes within sub-jects. Therefore, conclusions arising from this work are ade- Is it useful to prolong culture after thawing quately supported by the statistical analysis plan. Finally, in order to observe initial blastomere proliferation? although pregnancy and live birth rates are commonly usedoutcomes, the first is still an intermediate measure and does Although some previous reports seemed to demonstrate that not discriminate between implantation of one or more embry- post-thawed cleaving embryos with observed blastomere pro- os, while the second returns a result in terms of oocyte pick-up liferation hold a higher developmental potential than non- or embryo transfer procedures and not in relation to each cleaved ones , ], we found no evidence of increased transferred embryo. As this study aims to center on individual implantation and live birth rates per embryo after transferring embryo developmental potential following two different post- only embryos where blastomere proliferation had been ob- thaw culture durations and not on live birth per woman, served after a long post-thaw culture. These results contrast implantation rate and live birth rate per embryo were consid- with others reported for the same type of transfers, possibly ered to be more accurate and informative outcome measures.
due to the statistical analysis or to the non-elective approach of In fact, more important than the actual, unadjusted, values our FETs and to the marked influence of common biasing observed for implantation and live birth rate per embryo factors, such as age and number of embryos transferred, in the represented in Fig. are the adjusted differences between most recently reported data [Finally, the negative influence groups (Fig. which estimate the independent effect of that a prolonged post-thaw culture exerts on embryonic de- distinct post-thaw culture periods after controlling for six velopmental potential reinforces the concept that the inability potential confounding factors.
to develop in vitro is no evidence of developmental incompe-tence in vivo [Our results are in favor of such concept, Shorter post-thaw culture periods allow embryos to maintain as the outcome of post-thaw cleaved embryos and those higher implantation and developmental potential selected based only upon survival is similar. This indicatesthat the developmental potential of non-cleaving embryos Our results indicate that the probability of frozen-thawed could be better preserved if they would only be briefly ex- embryos to implant and develop to term is significantly posed to post-thaw in vitro conditions.
increased if embryos are only briefly cultured after thawing.
This observation highlights the environmental impact that Final considerations and conclusion post-thaw culture length has on embryo developmental po-tential. Negative influences of culture upon embryo devel- We all agree that the best outcome achieved after embryo opment have been pointed out and, although embryo culture cryopreservation will result from embryos with a high score aims to mimic the fallopian tube and uterine environment, it before freezing, that not only retain their initial blastomere always implies induction of some related stress [–].
number, but above all retain their developmental competence It thus seems reasonable to propose that frozen-thawed and that must end up resuming mitotic activity in the uterine embryos may be less able to cope with, and adapt to, sub- cavity. Our results suggest that a prolonged post-thaw culture optimal environments such as the currently available culture period can be causing a decrease in the implantation and systems ]. By reducing the post-thaw culture duration, developmental potential of thawed cleavage stage embryos.
there will be a decrease of culture-related stress, better The present study raises the interesting proposal that it may preserving embryonic developmental potential.
not be desirable to actually see the resumption of mitotic As previously reported, many factors have been pointed activity in some blastomeres of thawed embryos, because that out as influencing post-thaw outcome, but their relative con- usually requires a long post-thaw culture period. This hypoth- tributions are variably accepted ]. When comparing ART esis now demands further validation through prospective ran- outcomes, maternal age at OPU and number of embryos domized studies.
transferred are usually accepted as the two most importantcauses of bias in intra- and inter-assay comparisons. At freez-ing, the two most referred embryological factors are embryo Conflict of interests The authors declare that they have no conflict grading and embryo developmental day , ], while of interest.
after thawing, the two most discussed embryological factorsare blastomere cryosurvival , ] and the visualization of This article is distributed under the terms of the Crea- blastomere mitotic resumption ]. In the light of the tive Commons Attribution License which permits any use, distribution, J Assist Reprod Genet and reproduction in any medium, provided the original author(s) and 12. Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Tesquier L, the source are credited.
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Water Air Soil PollutDOI 10.1007/s11270-011-0864-z Characterization of Swine Wastewater by ToxicityIdentification Evaluation Methodology (TIE) C. Alejandra Villamar & Teresa Cañuta &Marisol Belmonte & Gladys Vidal Received: 25 January 2011 / Accepted: 13 June 2011 # Springer Science+Business Media B.V. 2011 Abstract Since swine wastewater is used by farmers (around 100%) and total nitrogen (95.5%). This