Leukaemia inhibitory factor modulates the differentiation of granulosa cells during sheep in vitro preantral to antral follicle development and improves oocyte meiotic competence.

In vitro follicle development from cryopreserved ovarian tissue could become an invaluable assisted reproduction technology for women with early ovarian failure. The challenge lies in producing, from small follicles present in the ovarian cortex, high-quality mature oocytes able to sustain embryo development. In vivo, an optimal combination of hormones and other factors coordinates the development of follicles and their enclosed oocyte. We have investigated the effect of the leukaemia inhibitory factor (LIF) cytokine, alone or in combination with FSH, on sheep in vitro follicle development from the preantral stage onwards. LIF did not alter follicle growth or antrum formation, but it modulated the differentiation of granulosa cells, as revealed by decreased production of anti-Müllerian hormone and abolished FSH-induced stimulation of oestradiol secretion. This modulatory role was also reflected in the abundance of mRNA from 35 genes, analysed by reverse-transcription coupled to microfluidic quantitative PCR. LIF stimulated or at least maintained the expression of genes involved in the dialogue between the oocyte and granulosa cells, through gap junctions (GJA4 encoding connexin 37) or paracrine signalling (Bone morphogenetic protein 15, KIT ligand and their receptors). Finally, the presence of both LIF and FSH during follicle growth strongly improved oocyte meiotic competence: most oocytes (56%) underwent subsequent nuclear maturation, a significant increase compared with their counterparts from follicles of similar size (550-900 µm) cultured with FSH only (28%) or developed in vivo (9%). Their ability to sustain embryo development remains to be evaluated. Combined supplementation with FSH and LIF certainly merits investigation with human follicles.

Extended embryo culture is effective for patients of an advanced maternal age.

The aim of this study was to determine the effectiveness of extended embryo culture in advanced maternal age (AMA) patients (37-43 years). In this retrospective analysis, 21,301 normally fertilized zygotes from 4952 couples were cultured until the blastocyst stage. Blastocyst development, including kinetics and morphology, transfer rate, implantation and live birth rates, were measured. In AMA patients, the blastocyst rate was significantly decreased as compared to that in younger women. On day 5, blastocysts underwent growth retardation in AMA patients, which was highlighted by a decreased rate of full/expanded blastocysts. Organization of the cells (trophectoderm and inner cell mass) was unaffected by age. However, in AMA patients, a 'good' morphology blastocyst had a decreased probability to implant compared with an 'average' morphology blastocyst in younger women. While the rates of blastocyst transfer and useful blastocysts were similar to younger patients, in AMA patients, both implantation and live birth rates were significantly reduced. Our results support the idea that extended embryo culture is not harmful for AMA patients. However, embryo selection allowed by such culture is not powerful enough to avoid chromosomal abnormalities in the developed blastocysts and therefore cannot compensate for the effect of a woman's age.

Bisphenol S is present in culture media used for ART and cell culture.

STUDY QUESTION: Do plastic laboratory consumables and cell culture media used in ART contain bisphenols? SUMMARY ANSWER: The majority of human embryo culture media assessed contained bisphenol S close to the nanomolar concentration range, while no release of bisphenols by plastic consumables was detected under routine conditions. WHAT IS KNOWN ALREADY: The deleterious effect of the endocrine disruptor bisphenol A (BPA) on female fertility raised concerns regarding ART outcome. BPA was detected neither in media nor in the majority of plastic consumables used in ART; however, it might have already been replaced by its structural analogs, including bisphenol S (BPS). STUDY DESIGN, SIZE, DURATION: Seventeen plastic consumables and 18 cell culture and ART media were assessed for the presence of bisphenols. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ten different bisphenols (bisphenol A, S, AF, AP, B, C, E, F, P and Z) were measured using an isotopic dilution according to an on-line solid phase extraction/liquid chromatography/mass spectrometry method. MAIN RESULTS AND THE ROLE OF CHANCE: While the plastic consumables did not release bisphenols under routine conditions, 16 of the 18 cell culture and ART media assessed contained BPS. Six media exhibited BPS concentrations higher than 1 nM and reached up to 6.7 nM (1693 ng/l). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Further studies are required to investigate a greater number of ART media to identify less potentially harmful ones, in terms of bisphenol content. WIDER IMPLICATIONS OF THE FINDINGS: As BPS has already been reported to impair oocyte quality at nanomolar concentrations, its presence in ART media, at a similar concentration range, could contribute to a decrease in the ART success rate. Thus far, there has been no regulation of these compounds in the ART context. STUDY FUNDING/COMPETING INTERESTS: This study was financially supported by the 'Centre-Val de Loire' Region (Bemol project, APR IR 2017), INRAE, BRGM, the French National Research Agency (project ANR-18-CE34-0011-01 MAMBO) and the BioMedicine Agency (Project 18AMP006 FertiPhenol). The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Clinical outcome after insemination with donor sperm in patients with poor results in ICSI cycles.

The introduction of intracytoplasmic sperm injection (ICSI) provided an effective treatment for infertile couples whose infertility was attributed to male factors. However, some of them face poor results after ICSI and subsequently use artificial insemination with donor sperm (AID). Only a few studies have reported on the clinical outcome of AID cycles after previous failed ICSI cycles, with contrasting results. The results reported here involve a cohort of 47 couples undertaking 175 AID cycles after 120 failed ICSI cycles for various reasons. Couples were allocated to two groups according to the availability of top quality embryos (TQE) in ICSI cycles. In our series, AID was successful for couples with and without TQE previously transferred in ICSI cycles, the live birth rate (LBR) per cycle being 20.0% and 13.3%, respectively. However, couples with TQE tended to succeed more rapidly than couples with poor quality embryos, with a higher cumulative LBR (68.0% versus 54.5%, respectively). These findings demonstrate that even couples with a history of unsuccessful ICSI cycles because of poor embryo quality are able to achieve high LBR after AID cycles. However, such couples have a lower cumulative LBR and are required to be more patient to achieve parenthood.

Semen variation in a population of fertile donors: evaluation in a French centre over a 34-year period.

Although it has been suspected that there is a decrease in semen quality over time, the results reported to date remain debatable because of methodological issues. The aim of the study reported here was to investigate the evolution of semen quality over time in a population of 1114 fertile candidates for sperm donation at CECOS, Tours, between 1976 and 2009. We investigated semen volume, sperm concentration, progressive motility, vitality, percentage of normal forms and multiple abnormalities index of the first ejaculate in this population. We did not find a decline in semen volume, whereas we observed a significant decrease in total sperm count (from 443.2 million in 1976 to 300.2 million in 2009), motility (from 64% in 1976 to 49% in 2009) and vitality (from 88% to 80%). Moreover, a significant decline in the percentage of normal forms was noted between 1976 and 1997 (from 67% to 26%) with a steady rise in the multiple abnormalities index between 1998 and 2009 (from 1.19 to 1.65). This study involving a population of fertile men from a restricted area revealed various degrees of decline in semen parameters over a period of 34 years. These findings will have to be compared with findings in other geographical areas.

[Single pregnancy outcome after blastocyst transfer: comparison with cleavage stage embryo transfers]. / Devenir des grossesses uniques après transfert au stade blastocyste : comparaison avec les transferts précoces.

OBJECTIVES: It has been clearly shown that transfer of fresh blastocysts was efficient in in vitro fertilization cycles. However, only few data have been published about pregnancy outcome and health of children born. This study including only single pregnancies compared pregnancy, delivery and neonatal outcomes after blastocyst transfers in comparison with early cleavage stage embryo transfers (Day 2). PATIENTS AND METHODS: A retrospective study has been performed at the hospital of Tours from January 2002 to June 2009. Considering only primiparous women with similar ages, the outcome of 1574 single pregnancies issued from blastocyst transfers (n=588) or early cleavage stage transfers (n=959) has been compared. Data have been collected from FIVNAT forms, filled in by voluntary couples. RESULTS: The rate of return of FIVNAT forms was 97.6%. The pregnancy and delivery outcomes, sex ratio, birth weight, health of newborns were similar between both groups. DISCUSSION AND CONCLUSION: Regarding pregnancy, obstetric and delivery outcomes after blastocyst transfers, the literature data remain poor, much debated, and mixing often single and multiple pregnancies. The results of this study seem reassuring but have to be confirmed by further studies.

Treating women under 36 years old without top-quality embryos on day 2: a prospective study comparing double embryo transfer with single blastocyst transfer.

BACKGROUND Embryologists currently face a challenge when counselling patients regarding the stage and the number of embryos to transfer when no top-quality embryos (TQE) are available. METHODS The aim of this study was to evaluate the efficacy of single blastocyst transfer (SBT) in comparison with the transfer of two cleavage-stage embryos in women under 36 years old. A total of 450 women under 36 years undergoing their first or second IVF treatment who had no TQE on Day 2 were included in this prospective study. Couples were assigned to either a SBT or a double cleavage-stage embryo transfer (DET). The clinical end-points monitored were rates of implantation, delivery and multiple deliveries. RESULTS The rate of transfer was significantly lower for couples assigned to the SBT group compared with the DET group (88 versus 100%, respectively, P < 0.001) while the delivery rate per oocyte retrieval was similar in both groups (26.7%). By contrast, the rate of multiple deliveries was significantly lower in the SBT group compared with the DET group (3.3 versus 23.3%, respectively, P < 0.01). Blastocyst cryopreservation was twice as high in the SBT group compared with the DET group (39 versus 18%, respectively, P < 0.001). CONCLUSIONS These findings show the value of extended embryo culture for couples without TQE. In such situations, delaying embryo transfer in order to select a single blastocyst with the highest potential for implantation can reduce the number of multiple pregnancies. Furthermore, our results demonstrate that extended culture allows blastocyst cryopreservation from embryos not available for Day 2 cryopreservation.

Sperm morphology assessment using David's classification: time to switch to strict criteria? Prospective comparative analysis in a selected IVF population.

The aim of this study was to compare assessment of sperm morphology by using David's classification (DC), a method of manual analysis most common in France, with a computer-assisted method (Integrated Visual Optical System) based on the strict criteria (CASA SC) for their ability to predict fertilization in a selected in vitro fertilization (IVF) population. A total of 120 couples engaged in IVF protocols were prospectively included in the study. To focus mainly on sperm morphology, couples were excluded in cases of abnormalities of sperm concentration and/or motility and immunological factors and when a low number of oocytes were collected. Sperm morphology analysis was performed on the day of oocyte retrieval by the same trained biologist. Our results showed a moderate correlation between the two techniques (r = 0.49). The DC sperm morphology analysis was less indicative of fertilization than CASA SC (r = 0.07, p = 0.47 vs. r = 0.22, p = 0.014). Using receiver-operating characteristics analysis, we showed that DC was not discriminating in the prediction of fertilization (AUC = 0.572). DC seemed less appropriate for the prediction of fertilization success or failure. In contrast, with CASA SC, the previously determined cut-off value of around 14% was confirmed (AUC = 0.735, cut-off = 6%). Our results argue in favour of the replacement of DC by SC to tend towards worldwide standardization.

Does early morphology provide additional selection power to blastocyst selection for transfer?

Routine early developmental parameters are widely used in IVF centres to evaluate embryo development and fresh single-blastocyst transfer currently seems superior to single-embryo transfer. Would early morphological parameters help to choose the single blastocyst to be transferred, thereby improving the chances of implantation and live birth rate? This prospective observational study analysed the individual outcomes of 2617 embryos from 511 IVF couples scheduled for a single-blastocyst transfer. Embryo and blastocyst scores were constructed. There was a clear relationship between the kinetics and morphology of blastocysts and further implantation and live birth rate. There was a limited predictive value of embryo score with regard to blastocyst development and growth kinetics. Implanted and non-implanted blastocysts showed similar embryo scores. Thus usual morphological parameters on days 1 and 2 seem to have no additional value in indicating the right blastocyst to transfer. Non-invasive approaches might be helpful to increase the chances of implantation in the future.

[Which assisted reproduction technique as a function of sperm morphology?]. / Choix de la technique d'Assistance médicale à la procréation en fonction du spermocytogramme.

Semen analysis is routinely part of the evaluation of male fertility and includes sperm morphology. How sperm morphology can be a diagnostic tool to decide which ART to apply for infertile couples (insemination, classical IVF or ICSI)? Teratozoospermia has been reported to be one of the most relevant semen parameters of clinical interest compared with sperm concentration and sperm motility. However, the true potential of this parameter has some limitations because of several methodological factors: differences in the classification systems, influence of the staining method used, subjective nature of evaluation of sperm morphology, regular decreasing of the WHO threshold. According to Tygerberg strict criteria that is the most frequently used classification worldwide, poor fertilization has been reported when the rate of normal forms was less or equal to 4%, suggesting to perform ICSI. However, influence of sperm morphology on fertilization outcome remains much debated. Regarding David's classification, the threshold of 30% does seem helpful to discriminate in the prediction of fertilization success and should probably be reconsidered.

[Non invasive assessment of embryo quality: proteomics, metabolomics and oocyte-cumulus dialogue]. / Approches non invasives de l'embryon : protéomique, métabolomique, dialogue ovocyte-cumulus.

Preimplantation embryo development is one of the key features with implantation itself to achieve a pregnancy. Assisted Reproductive Technologies both in human and animal have improved our knowledge on these events, although it remains elusive to predict embryo potential to give a baby. Among various ways to define embryo viability, noninvasive approaches get a serious advantage linked to the final transfer of the embryo. Techniques devoted to characterize the embryo secretome using proteomic or metabolomic approaches may be non invasive. Based on a direct identification of products of the embryo metabolism or an assessment of profile(s) related with embryo viability, they have greatly improved their sensitivity to allow their use in clinical embryology, once validated. Oocyte-cumulus dialogue, as a key factor for oocyte competence to meiosis and embryo development, was particularly concerned with both genomic and proteomic assessment of cumulus cells. While it is not possible to designate at the time being which among these approaches will be robust and cost-efficient enough to help routinely the clinical embryologist in assisted reproductive techniques (ART), one can predict that our ability to select the "right" embryo will combine morphological criteria already available with validated biomarkers.

Serum antimüllerian hormone is not predictive of oocyte quality in vitro fertilization.

OBJECTIVES: The assessment of the ovarian reserve is mandatory in women undergoing assisted reproduction. Antimüllerian hormone (AMH) produced by granulosa cells from preantral and early antral follicles, is a promising indicator of ovarian reserve. However, few studies have evaluated the predictive value of AMH on oocyte quality. MATERIAL AND METHODS: A retrospective study was undertaken at the Bretonneau University Hospital of Tours. A total of 559 women undergoing in vitro fertilization treatment between January 2007 and December 2007 were included in the study. Serum AMH levels were determined by using an ultrasensitive ELISA test. Total number of oocytes, rate of mature oocytes, fertilization rate, embryo quality and clinical pregnancy rate were recorded. RESULTS: Serum AMH was significantly lower in groups of patients with few oocytes collected. However, serum AMH was not predictive of nuclear maturity of oocytes, fertilization rate and quality of early embryos. Additionally, low levels of AMH do not preclude clinical pregnancy, in in vitro fertilization. CONCLUSION: At the moment, serum AMH is a relatively predictive indicator of the ovarian reserve, in terms of quantity but not in terms of quality. Moreover, it is still not possible to determine serum AMH cut-off value to predict clinical pregnancy in IVF programmes.

Single Day 2 embryo versus blastocyst-stage transfer: a prospective study integrating fresh and frozen embryo transfers.

BACKGROUND: Whether extended culture allowing selection of embryos with high development potential has any advantage over cleavage-stage embryo transfer remains a matter of debate. Among the currently unsolved questions, the cumulative delivery rate resulting from fresh and frozen embryo transfers needs to be taken into account in both strategies. The aim of our study was, therefore, to compare the efficacy of single embryo transfer either on Day 2 or on Day 5/6 combining fresh and frozen embryo transfers. METHODS: A prospective study including 478 couples assigned on a voluntary basis to undergo elective single embryo transfer (eSET, n = 243) on Day 2 or single blastocyst transfer (SBT, n = 235) on Day 5/6 was performed. The primary outcome measurement was the cumulative delivery rate including fresh and frozen-thawed cycles in both groups. RESULTS: The delivery rate per cycle following fresh embryo transfer was significantly higher in the SBT group compared with the eSET group (P < 0.01). Conversely, frozen embryo and/or blastocyst transfers tended to result in a higher number of deliveries in the eSET compared with the SBT group. Altogether, the cumulative delivery rate per couple, including fresh and frozen embryo transfers, was similar between the two groups (37.9% versus 34.2% in the SBT and eSET groups, respectively). CONCLUSIONS: The observed cumulative delivery rates in this study do not allow us to take a position in favor of SBT or eSET. An improvement in blastocyst cryopreservation may change this attitude.

[Preimplantation embryo development: current status and perspectives in clinical embryology]. / Développement de l'embryon préimplantatoire : état actuel et perspectives en embryologie clinique.

Preimplantation embryo development is one of the key features with implantation itself to achieve a pregnancy. Assisted Reproductive Technologies (ART) both in human and animal have improved our knowledge on these events, although predicting embryo potential to give a baby remains elusive. However data from the last 10 years have allowed either to hierarchize the available parameters or to open some new perspectives to predict embryo developmental potential. Both kinetics and morphological parameters belong to noninvasive quality embryo assessment for many years, although recent data on microvideographic analysis and multivariate analysis led to reduce their biological meaning. Moreover, new technical detection of meiotic spindle birefringency or zona pellucida anisotropy has improved the oocyte quality assessment with a deep impact for countries with restrictive legislation. Beyond such morphological criteria, more functional approaches concerned the oocyte (embryo) or its environment. Direct transcriptomic analysis, while invasive and therefore experimental, brought important data on embryo "quality". However, noninvasive metabolomic or proteomic analysis of embryo media gave promising results as well as respirometry. The environment of the oocyte has focused a specific attention, either based on regulatory proteins or cytokines present in follicular fluid, or involving genes or proteins from cumulus cells, as oocyte-cumulus dialog is a key factor in oocyte maturation. Whereas it is not possible for the time being to predict which parameter(s) will be implemented routinely, all data obtained underline that the ability to develop and implant is not based on embryo superlatives (more rapid, expressing more genes or proteins, larger metabolites uptake) but rather on a quiet state, as claimed by Leese some years ago, where a lot of resources would not be mobilized by any stressful situation.

Gene expression in human cumulus cells: one approach to oocyte competence.

BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.

Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos.

BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.

Meiotic segregation in spermatozoa of a 45,XY,-14,der(18)t(14;18)(q11;p11.3) translocation carrier: a case report.

A 35-year-old male was found to have a 45,XY,-14,der(18)t(14;18)(q11;p11.3) karyotype during the investigations for a couple with infertility for 8 years. Two sperm samples were obtained and analysed in triple fluorescence in situ hybridization (FISH) with the D18Z1 and LSI IGH/BCL2 probes. The frequency of gametes exhibiting a normal or balanced chromosomal equipment was 87.26 and 90.97% in samples 1 and 2, respectively. No statistically significant difference was found between the results of meiotic segregation of both samples. These proportions are close to those observed among Robertsonian translocation carriers. They can probably be explained by the formation of trivalent in cis configuration during meiosis I between the derivative chromosome and the normal chromosomes 14 and 18, as in Robertsonian translocation carriers. These results suggest that the configuration adopted at pachytene strongly determines the segregation mode that will be preferentially followed during anaphase I.

Meiotic segregation analysis in spermatozoa of pericentric inversion carriers using fluorescence in-situ hybridization.

BACKGROUND: Pericentric inversions are structural chromosomal abnormalities resulting from two breaks, one on either side of the centromere, within the same chromosome, followed by 180 degrees rotation and reunion of the inverted segment. They can perturb spermatogenesis and lead to the production of unbalanced gametes through the formation of an inversion loop. METHODS: We report here the analysis of the meiotic segregation in spermatozoa from six pericentric inversion carriers by multicolour fluorescence in-situ hybridization (FISH) and review the literature. RESULTS: The frequencies of the non-recombinant products (inversion or normal chromosomes) were 80% for the inv(20), 91.41% for the inv(12), 99.43% for the inv(2), 68.12% for the inv(1), 97% for the inv(8)(p12q21) and 60.94% for the inv(8)(p12q24.1). The meiotic segregation of 20 pericentric inversions (including ours) is now available. The frequency of unbalanced spermatozoa varies from 0 to 37.85%. The probability of a crossover within the inverted segment is affected by the chromosome and region involved, the length of the inverted segment and the location of the breakpoints. CONCLUSIONS: No recombinant chromosomes were produced when the inverted segment involved <30% of the chromosome length (independent of the size of the inverted segment). Between 30 and 50%, few recombinant chromosomes were produced, inducing a slightly increased risk of aneusomy of recombination in the offspring. The risk of aneusomy became very important when the inverted segment was >50% of the chromosome length. Studies on spermatozoa from inversion carriers help in the comprehension of the mechanisms of meiotic segregation. They should be integrated in the genetic exploration of the infertile men to give them a personalized risk assessment of unbalanced spermatozoa.

[Sequential assessment of individually cultured embryos in IVF]. / Suivi individuel des embryons en FIV.

OBJECTIVE: It is a challenge for IVF centers to propose a method to select the most viable embryo to transfer, thereby minimizing the risk of multiple births. In this study, a prospective investigation was made to determine if non-invasive developmental markers on day 1 combined to conventional evaluation on day 2 can predict in vitro blastocyst development. PATIENTS AND METHODS: A total of 4190 individually cultured embryos from patients undergoing IVF/ICSI treatment at the Tours University Hospital Center from January 2002 to December 2004 were included. Individual embryos were cultured in sequential media in microdrops under mineral oil from j1 to j5/j6 allowing to record their sequential growth until the blastocyst stage. RESULTS: The results showed a significant positive relationship between pattern 0 zygote, early cleavage, 4 cells embryos with < 20% fragmentation on day 2 and the rate of blastocyst development on day 5 (P < 0.05). In our hands, zygote pattern does not bring additional benefit to better select embryo. DISCUSSION AND CONCLUSION: Zygote and early cleavage assessments on day 1, morphological appearance on day 2 are some other parameters related individually to blastocyst development on days 5 and 6. These parameters can be used collectively to establish a predictive in vitro sequential embryo assessment model for routine use in IVF clinics.