Effect of oocyte vitrification on embryo quality: time-lapse analysis and morphokinetic evaluation

Effect of oocyte vitrification on embryo quality: time-lapse analysis and morphokinetic evaluation Ana Cobo, Ph.D., Aila Coello, Ph.D., Jose Remohí, M.D., Jose Serrano, B.Sc.T., Jose Maria de los Santos, Ph.D., and Marcos Meseguer, Ph.D. Instituto Valenciano de Infertilidad Valencia, INCLIVA–University of Valencia, Valencia, Spain

Objective: To analyze whether oocyte vitrification may affect subsequent embryo development from a morphokinetic standpoint by means of time-lapse imaging. Design: Observational cohort study. Setting: University-affiliated private IVF center. Patient(s): Ovum donation cycles conducted with the use of vitrified (n ¼ 631 cycles; n ¼ 3,794 embryos) or fresh oocytes (n ¼ 1,359 cycles; n ¼ 9,935 embryos) over 2 years. Interventions(s): None. Main Outcome Measure(s): Embryo development was analyzed in a time-lapse imaging incubator. The studied variables included time to 2 cells (t2), 3 cells (t3), 4 cells (t4), 5 cells (t5), morula (tM), and cavitated, early, and hatching blastocyst (tB, tEB, tHB) as well as 2nd cell cycle duration (cc2 ¼ t3  t2). All of the embryos were classified according to the hierarchic tree model currently used for embryo selection. The analyzed variables were compared with the use of analysis of variance or chi-square and included 95% confidence intervals (CIs). Result(s): The embryos that originated from vitrified oocytes showed a delay of 1 hour from the first division to 2 cells (t2) to the time of blastulation (tB). The embryos that originated from vitrified oocytes showed a delay of 1 hour from the 1st division to 2 cells (t2) to the time of blastulation (tB) (P< .05). The proportions of embryos allocated to categories A–E in the hierarchical tree were similar between groups. No differences in implantation rates between the fresh (51.3% [95% CI 47.1%–55.7%]) and vitrified (46.4% [95% CI 38.4%–54.4%]) groups were found. Conclusion(s): The embryo quality of vitrified oocytes was not impaired: cc2, quality according to our hierarchic morphokinetic model, and implantation rates were similar between fresh and vitrified oocytes. However, morphokinetic differences were observed from t2 to tB. Our main study limitation was the retrospective nature of the analysis, although a large database was studied. (Fertil SterilÒ 2017;108:491–7. Ó2017 by American Society for Reproductive Medicine.) Key Words: Vitrification, morphokinetics, time-lapse, oocyte, embryo Discuss: You can discuss this article with its authors and with other ASRM members at https://www.fertstertdialog.com/users/ 16110-fertility-and-sterility/posts/17978-23991

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he possibility of successful female gamete cryopreservation is a remarkable milestone in contemporary assisted reproductive technology. Ovum donation programs have been major beneficiaries of the establishment of efficient egg-banking. Oocyte cryostorage results are very useful for overcoming the most common

drawbacks involved with the use of fresh donations, such as synchronization between donors and recipients and long waiting lists, and make the process safer, given the possibility of saving the quarantine period. Egg banking has been possible thanks to vitrification providing high success rates (1, 2). In the past decade, the use of donors’ vitrified

Received March 6, 2017; revised June 16, 2017; accepted June 20, 2017. A.Cobo has nothing to disclose. A.Coello has nothing to disclose. J.R. has nothing to disclose. J.S. has nothing to disclose. J.M.d.l.S. has nothing to disclose. M.M. has nothing to disclose. Supported by the Spanish Ministry of Economy and Competitiveness (PI14/00523) through an Instituto de Salud Carlos III program and by a grant from Fertility Innovation, Merck Serono. Reprint requests: Ana Cobo, Ph.D., Instituto Valenciano de Infertilidad (IVI), University of Valencia, Pl. Policía Local 3, Valencia, Spain (E-mail: [email protected]). Fertility and Sterility® Vol. 108, No. 3, September 2017 0015-0282/$36.00 Copyright ©2017 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2017.06.024 VOL. 108 NO. 3 / SEPTEMBER 2017

oocytes has gradually increased, and it can be stated that egg banking in ovum donation programs is currently a frequent approach (2). Given the positive results, oocyte cryopreservation has been conveyed to other applications, mainly for women who wish to preserve fertility (3). The suitability of oocyte vitrification has been established thanks to the evaluation of survival rates, embryo development parameters, and implantation, pregnancy, and live birth rates. These outcomes have been typically compared between fresh and vitrified oocytes or between slow freezing and vitrification. Similar embryo development 491

ORIGINAL ARTICLE: ASSISTED REPRODUCTION has been shown in embryos that originate from fresh versus vitrified oocytes in a sibling cohort study (4), and the clinical validation of using vitrified oocytes for egg donation has been demonstrated in a large randomized controlled clinical trial (5). Similar obstetrical and perinatal outcomes of the babies conceived with the use of vitrified versus fresh oocytes has been recently demonstrated in a large study that involved more than 2,000 infants (6). Classically, embryo quality evaluations have been based on morphologic criteria, which involves conventional static observations that are linked to specific time points and interobserver variations. Embryo development is a dynamic process, and several critical stages can go unnoticed with the use of traditional morphologic assessments. As previously demonstrated, the time-lapse imaging of cell division kinetics offers a far more accurate analysis of embryo development and provides detailed information of the dynamic morphology in each cell-division step (7, 8). Relevant parameters, such as multinucleation and fragmentation, critical abnormal cleavage patterns, and morphokinetics can (9) be seen or evaluated only with the use of time-lapse technology (10–14). Very little is known about the morphokinetics of the embryos generated from vitrified oocytes. Vitrification consists in the solidification of an aqueous solution in the absence of ice crystals. During the process, cells are subjected to high osmotic stress and most intracellular water content is replaced with permeable cryoprotectants. On warming, cells rehydrate and cryoprotectants are removed. Whether all of these physicochemical changes cause any alteration in embryo morphokinetics is still not well known. The only report on this topic has been attempted with the use of the embryo kinetics of fresh and sibling vitrified-warmed oocytes, limited to a 44-hour time-lapse analysis on 168 fertilized sibling vitrified-warmed oocytes (15). Although no differences in clinical outcomes and embryo morphology have been observed in previous studies that have compared fresh and vitrified oocytes (4, 5), the time-lapse imaging of embryos from vitrified oocytes can help to elucidate whether vitrification can cause subcellular effects that are able to alter cell division dynamics. The purpose of the present study was to evaluate the effect of oocyte vitrification on the morphokinetic parameters of embryos generated after the vitrification and warming procedures compared with embryos developed from fresh oocytes in ovum donation cycles, with the use of the largest sample size reported to date and covering all embryo development stages.

MATERIALS AND METHODS Study Design and Population This was an observational 2-year cohort study approved by the Institutional Review Board that governs the clinical use of IVF procedures for research at the Instituto Valenciano de Infertilidad (ref. 1511-VLC-062-AC, 111/11/2015). The study included 1,359 ovum donation (OD) cycles (n ¼ 9,936 embryos) conducted with the use of fresh oocytes, and 631 492

OD cycles (n ¼ 3,794 embryos) carried out with the use of vitrified oocytes.

Protocol for Donors All of the involved donors fulfilled our inclusion criteria. The controlled ovarian stimulation (COS) protocols used in our center for donors are described elsewhere (1). In the GnRH agonist protocols, triggering was performed with the use of 250 mg recombinant hCG (rhCG; Ovitrelle; Serono). Alternatively, the flexible GnRH antagonist protocol was used as follows: COS was initiated on day 2–3 after bleeding with the use of 150 or 225 IU/d recombinant FSH (Gonal-F, Merck-Serono; or Puregon, MSD), combined with 75 IU/d hMG (Menopur; Ferring Pharmaceuticals). Doses were adjusted to ovarian response. Daily doses of 0.25 mg GnRH antagonist (ganirelix [Orgalutran; MSD] or cetrorelix [Cetrotide, Merck Serono]) were started when a follicle measuring >14 mm was observed. A single dose of the GnRH agonist (0.1 mg triptorelin; Decapeptyl; Ipsen Pharma) was administered to trigger final oocyte maturation when at least three follicles measuring >17.5 mm or one follicle measuring >20 mm was observed. In some cases, triggering was performed with the use of 250 mg rhCG. Transvaginal oocyte retrieval was conducted 36 hours later. The retrieved metaphase II (MII) oocytes were vitrified 2 hours after ovum pick-up. Anonymous donors were matched with their recipients according to phenotype and blood groups, and the assignment of fresh or vitrified donations depended on the availability of couples' suitable oocytes and/or patients’ preferences. Informed consent was obtained in all cases.

Endometrial Preparation for Oocyte Recipients As described elsewhere (1), women with ovarian function were first down-regulated in the luteal phase with the use of a single dose of GnRH agonist depot (3.75 mg Decapeptyl, Ipsen Pharm; or 3.75 mg Gonapeptyl Ferring). After menses, all subjects received oral E2 valerate (EV; 6 mg/d Progynova; Schering). Approximately 10–15 days after initiating EV, serum E2 and P levels and endometrial thickness were measured. Administration of micronized P (800 mg/d vaginally; Progeffik; Effik Laboratories) was initiated on the day after oocyte donation. If pregnancy was achieved, administration of EV and P was maintained until gestation week 12.

Oocyte Vitrification Protocol Oocytes were denuded 2 hours after oocyte retrieval. Fresh oocytes were denuded and microinjected 4 hours after oocyte retrieval. The MII oocytes were vitrified immediately after the nuclear maturity evaluation, by checking for the presence of the first polar body. All of the materials and tools for vitrification were obtained from Kitazato. The Cryotop method followed for oocyte vitrification has been described elsewhere (1, 16, 17). Specifically, after 12 minutes of stepwise equilibration in a mixture of 15% (v/v) ethylene glycol and dimethylsulfoxide in buffer media supplemented with hydroxypropyl cellulose (17), oocytes were exposed to a vitrification solution by maintaining the same mixture of VOL. 108 NO. 3 / SEPTEMBER 2017

Fertility and Sterility® cryoprotectants, but double concentrated (30% v/v), for 50– 60 seconds. Loading took place within the next 10 seconds by placing oocytes on the device contained in the minimum volume. Immediate plunging into liquid nitrogen induced vitrification. Four oocytes (maximum) were loaded per Cryotop. Oocytes were stored in vapor tanks (V1500-AB Isothermal Freezer; CBS) for a variable storage time (18). During warming, cryoprotectants were diluted by subjecting oocytes to a hyperosmolar solution that contained 1.0 mol/L trehalose at 37 C (17). Dilution continued for 3 minutes at room temperature in a half-diluted solution that contained the same sugar. The warming procedure was completed with the use of two washes (one lasting 5 minutes, the other 1 minute) in buffer solution at room temperature.

Culture Conditions After warming, oocytes were placed under standard culture conditions at 5% CO2 and atmospheric O2 at 37 C for 2 hours before the intracytoplasmic sperm injection. Once injected, oocytes were placed in the individual wells of a preequilibrated Embryoslide (Vitrolife). Wells were filled with 20 mL of our regular culture medium (Cook), and the medium was changed on day 3 when embryos were cultured to day 5– 6 (CCM; Vitrolife). The time-lapse system (TMS) used for monitoring the morphokinetic parameters was the Embryoscope (Vitrolife). Images were acquired every 20 minutes on seven different focal planes for 72 hours, 120 hours, or 144 hours of culture.

Embryo Score, Time-Lapse Analysis, and Kinetic Parameters The morphology of the cultured embryos was assessed at 48 hours, 72 hours, 120 hours, and/or 144 hours after insemination/injection. The evaluated parameters included cell number, symmetry, and granularity, as well as type and percentage of fragmentation, presence of multinucleated blastomeres, and degree of compaction (19). Blastocysts were scored on day 5 or 6 depending on the expansion of blastocele cavity and on the integrity of both the inner cell mass and trophectoderm cells (19). Images of each embryo were retrospectively analyzed with the Embryoviewer external image analysis software (Unisense Fertilitech), in which all of the listed embryo developmental events were annotated together with the corresponding timing of events in hours after intracytoplasmic sperm injection (ICSI). The kinetic parameters recorded in this study have been described elsewhere by our group (20, 21). The exact timing for each embryo division was calculated in hours after microinjection. Time of cleavage was defined as the first time point where a complete separation between newly formed blastomeres was observed. The markers of development included: the 1st cell division that led to 2 cells (t2) and subsequently divisions that led to 3, 4, and 5 cells (t3, t4, and t5, respectively). The time to formation of morula (tM) and blastocele cavity (tB), to blastocyst expansion (tEB), and to initiate hatching (tHB) were also recorded. The 2nd cell cycle duration (cc2), i.e., VOL. 108 NO. 3 / SEPTEMBER 2017

the duration of the period spent as a 2-blastomere embryo (t3  t2), was also recorded. The percentage of embryos that followed the timing of the cleavage-stage divisions were also assessed, as proposed by Meseguer et al. (2011) (7). Accordingly, optimal timings were considered, e.g., t5 48.8–56.6 h, s2 (t4  t3) 0.76 h, and cc2 11.9 h, based on the quartiles of the cleavage timing encountered in their study. We also identified the embryos that fulfilled any of the following proposed exclusion criteria: uneven blastomere size at the 2-cell stage, multinucleation at the 4-cell stage, and abrupt division from zygote to the 3blastomere embryo (cc2 5 h) (Supplemental Fig. 1).

Morphokinetic Categories All embryos were classified according to the hierarchic tree model based on the correlations between the morphokinetic parameters and embryo implantation, as proposed earlier (7). When considering t5 and s2, all of the evaluated embryos were allocated to five categories with decreasing implantation potentials from grade A to grade E (7). The further inclusion of cc2 allocated all of the evaluated embryos to 10 subcategories from Aþ to E. In the present study, the two first levels of this grading procedure were evaluated with the use of t5, taken as the primary timing variable, and s2, taken as the secondary timing variable. If the t5 value fell within the optimal range, the embryo was graded as A or B; if it fell outside the optimal range, the embryo was graded as C or D. If both s2 and t5 fell within the optimal range, the embryo was graded as A; if t5 fell within the optimal range, but s2 was outside it, the embryo was graded as B. If s2 fell within the optimal range, but t5 was outside it, the embryo was graded as C. If both s2 and t5 were outside the optimal range, the embryo was graded as D. The embryos that fulfilled any of the three following criteria were designated as grade E: 1) uneven blastomere size in the 2-cell stage (UN2); 2) abrupt division from 1 to 3 cells or more (DC1–3); or 3) multinucleation in the 4-cell stage (MN4).

Embryo Transfer Embryo transfers were performed on day 3 or in the blastocyst stage with the use of abdominal ultrasound guidance. The vitrification and warming procedures of the surplus embryos were performed according to our standard vitrification protocol as explained in detail elsewhere (22). The procedures were similar to those described above for oocytes, except that equilibration was performed at only one time (not stepwise) for 10 minutes. All of the remaining procedures, such as the vitrification step, loading, storage, and warming/dilution, were performed as described for oocytes.

Clinical Outcome Implantation rate was defined as the number of gestational sacs detected by means of transvaginal ultrasound examination divided by the number of replaced embryos; clinical pregnancy rate was confirmed by the detection of an embryonic sac during a transvaginal scan R5 weeks after embryo 493

ORIGINAL ARTICLE: ASSISTED REPRODUCTION transfer (23); and ongoing pregnancy was defined as the presence of a gestational sac with fetal heart beat observed during a transvaginal scan at R12 weeks (24). Live birth rate was also included in the analysis.

Statistical Analysis The results were analyzed with the use of an analysis of variance test for the comparison of timings (h) and a c2 test for the comparison of proportions, and they included 95% confidence intervals (CIs). The statistical analysis was run with the use of the Statistical Package for Social Sciences 22.0 (SPSS).

RESULTS The baseline oocyte donor and recipient parameters are presented in Table 1. In all, 9,936 zygotes originated from fresh oocytes and 3,794 zygotes from vitrified oocytes, which were cultured and monitored in the TMS. The implantation and clinical pregnancy rates are presented for the fresh and vitrified donations according to day of transfer (day 3 and blastocysts) in Table 2.

Embryo Kinetics of Development and Insemination In general, the embryos that originated from vitrified oocytes showed a delay of 1 hour from the 1st division to 2 cells (t2) to the time of blastulation (tB) (P< .05; Table 3). No differences were observed in the time needed for the fresh versus the vitrified oocytes to achieve complete expansion or to initiate hatching (Table 3). The proportions of embryos falling into the previously defined optimal timings (t5 48.8–56.6 h; s2 0.76 h; cc2 11.9 h) for embryos originated from fresh and vitrified oocytes for t5 (36.4% vs. 33.7%, respectively), s2 (47.5% vs. 47.5%), and cc2 (46.4% vs. 35.7%) showed statistical

differences (P< .05). In addition, DC1–3, MN4, and UN2 were compared and showed significant differences (Fig. 1). However, the proportions of embryos allocated to categories A–E were similar between groups when ranked according to the hierarchic tree model (Supplemental Table 1; available online at www.fertstert.org). In addition, implantation rates in the embryo categories depending on oocyte origin were similar (Supplemental Fig. 2; available online at www.fertstert.org).

DISCUSSION The first live birth after oocyte cryopreservation was achieved almost three decades ago (25). However, routine oocyte cryopreservation has become possible relatively recently, thanks to the introduction of improved vitrification techniques. Not even 10 years have passed since the systematic application of egg banking was established in IVF clinics worldwide. To date, oocyte vitrification has had a positive impact on ovum donation programs by providing efficient clinical results, and by making the logistics and functioning of these programs easier. Concurrently, other possible applications of the technology have been explored in patients who use their own oocytes, with excellent outcomes (26, 27). Consequently, patient demand has increased, especially for elective fertility preservation (28). Egg-banking efficiency was first demonstrated in ovum donations in a randomized clinical trial that compared the outcomes achieved after using fresh and vitrified oocytes (5). A large study, that included nearly 3,500 ovum donation cycles (>40,000 vitrified oocytes) with the use of this technology for 6 years, has confirmed the consistency of outcomes (1). According to a very recent study, outcomes from using autologous oocyte vitrification and warming procedures are as good as for cycles that use fresh oocytes in infertile populations (27). No differences in obstetrical and perinatal outcomes in babies born from vitrified versus fresh oocytes have been also reported (6).

TABLE 1 Donors and recipients’ demographic and baseline characteristics and fertilization and blastocyst formation for recipients. Donor characteristic No. of donation cycles Age (y) Survival rate Recipient characteristic No. of subjects Age (y) Body mass index (kg/m2) Days of endometrial preparation MII inseminated by ICSI (mean/ recipient) Fresh sperm concentration Fresh sperm motility Fertilization rate (2PN) Blastocyst rate (day 5) Blastocyst rate (day 6)

Fresh donors 1,359 26.2 (25.9–26.5) Recipients of fresh oocytes 1,359 40.4 (40.1–40.6) 25.0 (24.7–25.3) 18.4 (18.0–18.8) 10.5 (10.4–10.7) 47.4 (46.6–48.2) 31.7 (30.6–32.8) 78.0% (77.3%–78.6%) 73.8% (72.7%–74.9%) 79.8% (78.8%–80.8%)

Vitrified donors 631 26.4 (26.2–26.8) 94.5% (93.8%–95.2%) Recipients of vitrified oocytes 631 40.5 (40.3–40.7) 21.0 (20.8–21.2) 17.0 (16.5–17.5) 10.1 (99.9–10.3) 46.9 (45.2–48.6) 31.4 (30.3–32.5) 77.1% (76.1%–78.1%) 70.2% (68.2%–72.2%) 80.9% (79.1%–82.65%)

Note: Data are expressed as mean or n (95% confidence interval). 2PN ¼ two pronuclei; ICSI ¼ intracytoplasmic sperm injection; MII ¼ metaphase II. Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

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TABLE 2 Clinical outcome according to day of embryo transfer (ET). Fresh donations Outcome No. of cycles No. of ETs Mean embryos transferred Implantation rate (95% CI) Clinical pregnancy rate (95% CI) Ongoing pregnancy rate (95% CI) Live birth rate (95%CI)

Vitrified donations

Day 3 ET

Blastocyst ET

Day 3 ET

Blastocyst ET

862 1,638 1.90 36.2% (33.8%–38.7%) 51.8% (48.6%–54.9%) 43.8% (40.7%–46.9%)

497 840 1.69 51.6% (48.1%–55.1%) 66.3% (62.3%–70.2%) 58.9% (54.7%–62.9%)

443 833 1.88 36.1% (32.3%–39.9%) 50.7% (45.9%–55.4%) 43.2% (38.5%–47.9%)

188 323 1.72 49.4% (43.2%–55.5%) 63.9% (56.9%–60.9%) 56.3% (49.0%–63.5%)

37.8% (34.6%–41.0%)

53.4% (49.0%–57.8%)

35.9% (31.4%–40.9%)

52.0% (44.9%–59.1%)

Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

Vitrification can be achieved in the laboratory as a result of a balance struck between various factors, including the concentration and type of cryoprotectants, cooling and warming rates, and the volume containing samples (29). During the process, cells osmotically dehydrate when they come into contact with the cryoprotectant mixture, and the intracellular water content is replaced with the permeable cryoprotectant (30). These agents reach the glassy state when the temperature drops at a very high speed. On warming, the process is reversed and cells rehydrate while cryoprotectants are diluted. Whether these temporary drastic changes in cytosol composition can cause any alteration in the morphokinetic patterns of the embryos generated after oocyte warming remains unknown. Oocyte vitrification may act as an external factor that modifies the timing of the embryo progress.

TABLE 3 Embryo kinetics of development according to the type of cycle (fresh donations and donations with the use of vitrified oocytes). Timing (h) t2 t3 t4 t5 tM tB tEB tHB cc2 s2

Group

n

Mean (h)

95% CI

P value

Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified Fresh Vitrified

9,935 3,793 9,746 3,697 9,546 3,585 8,789 3,141 2,594 779 1,938 580 1,045 325 164 59 9,746 3,697 9,546 3,585

27.7 28.7 37.8 38.9 40.2 41.4 50.5 51.7 86.6 88.5 103.4 104.5 114.4 114.8 114.9 116.9 10.2 10.2 2.6 2.9

27.6–27.8 28.5–28.9 37.6–37.9 38.5–38.9 40.1–40.3 41.2–41.7 50.3–50.7 51.4–52.1 86.1–87.1 87.5–89.4 103–103.9 103.7–105.4 113.8–114.9 113.7–115.9 113.4–116.4 113.8–120 10.1–10.3 10.0–10.4 2.5–2.6 2.7–3.1

< .01 < .01 < .01 < .01 < .01 .016 .466 .196 .901 < .01

Note: t2 ¼ time to 2-cell embryo; t3 ¼ time to 3-cell embryo; t4 ¼ time to 4-cell embryo; t5 ¼ time to 5-cell embryo; tM ¼ time to morula; tB ¼ time to blastocyst; tEB ¼ time to expanded blastocyst; tHB ¼ time to blastocysts initiating hatching; cc2 ¼ t3  t2; s2 ¼ t4  t3. Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

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As far as we know, the entire body of literature that has evaluated oocyte vitrification is composed studies that have analyzed clinical outcomes or embryo quality with the use of the only tool that has been available to us until quite recently: embryo morphology. Although morphology is a recognized embryo quality evaluation parameter, lately morphokinetics has proven to be a very powerful tool, because subtle differences between individual embryos can be detected and it provides a much more objective measure of embryo development (9). The exact timing of each cell division, and the optimal ranges for the most discriminative kinetic parameters (t5, s2 and cc2), which correlated with a significantly higher probability of implantation, have been detected (7) and validated in retrospective and prospective clinical studies (10, 11). In the present study, we found some differences in the kinetics of embryos when they originated from vitrified oocytes. These differences were statistically significant and revealed a delay of 1 hour in each cell division until the early blastocyst stage in the vitrification group. One very interesting finding was cc2 was exactly the same between groups. This parameter was found to be one of the most predictive parameters of quality as described by Meseguer et al. (7). This is a relevant fact because DNA replication occurs during this period, and its duration is undisturbed in the embryos that come from vitrified oocytes. Therefore, we venture to state that the embryos in the vitrification group are actually no slower than fresh ones, but they start cellular divisions later and, most importantly, take the same time for DNA replication. This could explain why previous studies have shown similar implantation rates and clinical outcomes in vitrified versus fresh oocytes (5, 27). According to the literature, the reasons that explain alterations in the timing for cell divisions rely mainly on chromosomal alterations, which may delay DNA replications (31, 32). Alterations in embryo metabolism due to suboptimal culture conditions also cause significant changes in the expression profiles of developmentally important genes of hypoxic responses (32, 33) In addition, altered timings for cell division can be caused by some intrinsic factors within the oocyte, sperm, or both (31). The differential timing for the cell divisions observed in the vitrification group did not seem to obey any of these reasons, because the culture 495

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

FIGURE 1

Percentage of embryos falling within the optimal ranges proposed for the three variables included in the algorithm proposed by Meseguer et al. (2011): t5 (time to 5 cells), s2 (t4  t3), and cc2 (t3  t2), depending on oocyte origin (fresh vs. vitrified). In addition, exclusion criteria variables were included: direct-cleavage embryos (DC1–3), multinucleated embryos in the 4-cell stage (MN4), and embryos with uneven 1st cleavage (UN2). All proportions were significantly different between fresh and vitrified (P<.05). Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

conditions were strictly controlled in the TMS for both fresh and vitrified embryos. Moreover, some evidence indicates that oocyte vitrification does not raise aneuploidy rates in the embryos generated later (33). Further evidence may support the notion that embryo metabolism is not disturbed after showing oocyte vitrification with similar metabolomics profiles in embryos after the use of vitrified or fresh oocytes (34). Another possible explanation for the different morphokinetic parameters in early cell divisions after vitrification is quite simple in that, after stopping all the cell processes and metabolic reactions, reactivating the machinery may be time consuming and might imply extra efforts for cells, which might involve a higher energy cost. Mitochondria are essential to produce the energy required to perform all coordinated cellular processes, e.g., programmed cell death or spindle formation (35). Mitochondrial dysfunction in oocytes has been correlated with embryo arrest in vitro (36). Alterations in mitochondrial polarity after slow oocyte freezing have been associated with defects in Ca2þ signaling after insemination, whereas no changes in adenosine triphosphate (ATP) levels have been detected in thawed oocytes (37). Although changes in mitochondrial polarity (38) and ATP level alterations (39) are observed after oocyte vitrification, they seem to be temporary and are followed by a spontaneous recovery to the levels observed in fresh oocytes after 3–4 hours of culture. Furthermore, a very recent study has shown that vitrification affects the redox state toward oxidation, even though mitochondrial distribution and polarity are not affected by vitrification (40). It is worth mentioning that most of these studies were performed with the use of failed-to-fertilize or in vitro–matured oocytes, and it is difficult to extrapolate the results to fresh, i.e., not aged, MII oocytes (40). The authors have suggested including antioxidant agents in vitrification solutions to 496

later evaluate the redox state. It might also be interesting to include an evaluation of the morphokinetic parameters to rule out any possible effects of altered redox state and mitochondrial function on the delay in the timings for cell division observed in our study. We could hypothesize that diminished mitochondrial activity after oocyte vitrification could be responsible for the delay in the timings for cell division observed herein. The previously observed reversibility of alterations (39) backs the theory that vitrified oocytes need extra time to overcome the changes that take place. We could also speculate that a 1-hour delay of ICSI in the vitrification group may mitigate the delay in embryo divisions. However, the process develops normally once the machinery responsible for cell division is set in motion, as shown by the similar cell cycle durations and implantation rates. The observed distribution of all of the morphokinetic categories in the hierarchic tree was similar, and the implantation potential in each category did not differ between embryos from vitrified and fresh oocytes. This indicates that despite the delay in starting cell divisions, the embryos developed from vitrified oocytes had the same implantation potential as well as showed similar clinical and pregnancy rates. The smaller proportion of embryos in the vitrification group that fell within optimal ranges of cell division (t5, s2, cc2) was noteworthy. Nonetheless, these differences were not reflected when embryos were categorized according to the hierarchic tree model. In conclusion, although early embryo development in vitrified oocytes was delayed, the duration of the cell cycle, while the replication of DNA occurs is stable. Therefore, it remains unclear whether these alterations have consequences for the potential of vitrified oocytes, because no impact on implantation and clinical outcome was observed. VOL. 108 NO. 3 / SEPTEMBER 2017

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ORIGINAL ARTICLE: ASSISTED REPRODUCTION

SUPPLEMENTAL TABLE 1 Embryo grading according to the hierarchic tree. Morphokinetic category Oocyte

A

B

C

D

E

Fresh Vitrified

1,404 (16.0%) 498 (15.9%)

1,120 (12.7%) 344 (11%)

2,038 (23.2%) 712 (22.7%)

1,471 (16.7%) 615 (19.6%)

2,756 (31.4%) 972 (30.9%)

Fresh Vitrified

AL

AD

BL

BD

CL

CD

DL

DD

E

475 (5.4%) 201 (6.4%)

929 (10.6%) 297 (9.5%)

381 (4.3%) 122 (3.9%)

739 (8.4%) 222 (7.1%)

783 (8.9%) 397 (12.6%)

1,255 (14.3%) 315 (10.0%)

698 (7.9%) 370 (11.8%)

773 (8.8%) 245 (7.8%)

2,756 (31.4%) 972 (30.9%)

Note: No statistical differences were observed for all categories. Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

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SUPPLEMENTAL FIGURE 1

Morphokinetic decision tree algorithm used for embryo selection. t5 ¼ time to division to 5 cells; cc2 ¼ 2nd cell cycle, time from division to a 2blastomere embryo to division to a 3-blastomere embryo (t3  t2); s2 ¼ 2nd synchrony, duration of the division transition from a 2-blastomere embryo to a 4-blastomere embryo (t4  t3). The time of all events is expressed as hours after intracytoplasmic sperm injection. MN4 ¼ multinucleated embryo in the 4-cell stage. Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

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SUPPLEMENTAL FIGURE 2

Percentage of implanted embryos (implantation rate) depending on the embryo categories included in the algorithm proposed by Meseguer et al. (2011) and compared between fresh and vitrified oocytes. No statistical differences were observed in the implantation rates in each category depending on oocyte origin (P>.05). Cobo. Oocyte vitrification and morphokinetics. Fertil Steril 2017.

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