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Oocyte insemination techniques are related to alterations of embryo developmental timing in an oocyte donation model
Reproductive BioMedicine Online (2013) 27, 367– 375
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ARTICLE
Oocyte insemination techniques are related to alterations of embryo developmental timing in an oocyte donation model ˜oz a, ´s Garrido a,b, Blanca Gadea a, Manuel Mun Marı´a Cruz a, Nicola ´rez-Cano a, Marcos Meseguer a,b,* Inmaculada Pe a
Instituto Valenciano de Infertilidad, Alicante, Spain;
b
Instituto Universitario IVI Valencia, University of Valencia, Spain
* Corresponding author. E-mail address: [email protected] (M Meseguer). Dr Marcos Meseguer is a senior embryologist, scientific supervisor of Equipo IVI and an associate professor of the Masters in biotechnology from Valencia University. He performed a pre-doctoral fellowship in St Mary’s Hospital, Manchester University, UK. He received his PhD and the European Doctor Degree in obstetrics and gynaecology in 2002 and a Masters in research methods (design and statistics) in 2009. Dr Meseguer has received the prize paper from American Society of Reproductive Medicine, three times the Lalor Foundation International Award from the American Society of Andrology and Reproductive BioMedicine Online Robert Edwards prize paper in 2011. The primary areas of his research are embryology and male infertility.
Abstract Because of the different intrinsic characteristics of the classic IVF and intracytoplasmic sperm injection (ICSI) techniques,
the timing of zygote development can be influenced by the method of fertilization. However, there is no information about the relevance of the insemination procedure on embryo-quality parameters as measured through their developmental dynamics. The aim of this work was to determine if the insemination technique, IVF or ICSI, influences embryo developmental kinetics by examining 1203 embryos from 178 couples undergoing oocyte donation with IVF or ICSI. Using time-lapse information, this work calculated several developmental kinetic variables, from pronuclear fading (PNF) to expanded blastocyst, and also the proportion of optimal embryos in a best time range with a predicted higher implantation potential. Embryo development after ICSI was slightly faster than after IVF; however, when PNF, rather than time of insemination, was established as t0, the differences between the two procedures disappeared. The percentage of optimal embryos showed a trend towards higher values in IVF-derived embryos; however, the difference was not statistically significant. With these results and through the time-lapse monitoring system, it is concluded that it is the fertilization method which determines embryo developmental kinetics if insemination time is used as the starting point. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. KEYWORDS: embryo developmental kinetics, fertilization method, optimal embryos, time lapse
Introduction Little is known about the basic pathways and events of early human preimplantation embryo development, including
factors that would aid in predicting success or failure to develop. Consequently, to increase the chances of pregnancy through IVF, multiple embryos are often transferred to the uterus, despite the concern of well-documented
1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.rbmo.2013.06.017
368 adverse outcomes (Kalra and Barnhart, 2011). Therefore, in order to maximize the pregnancy rate without increasing multiple conceptions, it becomes paramount to develop a method of choosing the embryos that possess the greatest potential to implant. Routinely, embryo morphology and rate of development in culture have been the standard embryo selection techniques in IVF, since the available evidence indicates that a grading system based on these features is a satisfactory indicator of both blastocyst formation (Scott et al., 2000) and implantation potential (Ludwig et al., 2000). While basing the selection of transferable embryos on cell number and quality seems to provide some significant benefit (Cummins et al., 1986), pregnancy rates are still far from the highly theoretical limit of 100%. However, publications from the last decade have shown dramatic improvements compared with the early data and have reported that national success rates are on the rise (Schoolcraft et al., 2010; Shapiro et al., 2011). Furthermore, recently it was demonstrated that the success rate following assisted reproduction increases linearly each year when corrected for maternal age (Cohen et al., 2012). This latter report demonstrates that biological systems altered by technology will consistently improve over time and that this annual increment can be improved by faster incorporation of applied physical or digital systems. For example, the replacement of individual technical expertise with completely automated culture, growth and manipulation systems (Leung et al., 2011) may reduce variation and expedite progress toward a consistent and lasting 100% success rate. It is known that successful implantation, and as a result the pregnancy rate, depends on a competent embryo and a receptive endometrium with a synchronized dialogue between the maternal and embryonic tissues. However, pregnancy rates following IVF attempts remain low, where two out of three IVF cycles fail to result in a pregnancy and more than eight out of ten transferred embryos fail to implant (Kovalevsky and Patrizio, 2005). Given that many embryos, catalogued as displaying good prognosis using classical morphological assessment protocols, fail to implant, while other low-rated embryos are sometimes able to initiate and maintain a pregnancy, it is clear that the discriminative power of this analysis can be improved (Seli et al., 2010). A recognized shortcoming of the current methods of embryo evaluation is that they are static and only obtain information from very short intervals of time through embryo development, thus limiting a true assessment of the intricacies of morphological characteristics and cell number. McKiernan and Bavister (Bavister, 1995) highlighted this problem by stating that the examination of embryos at arbitrary time points during embryo development can be quite misleading with respect to categorizing the stage of development reached and ‘timelines’ of development. The importance of establishing the precise timing of development has been recognized in animal models, where embryos reaching the 8-cell stage in a defined time frame presented a greater ability to reach the blastocyst stage and a higher viability when transferred (Sakkas et al., 1998). Even in the human, the selection and transfer of early cleavage 2-cell embryos has consistently been shown to provide higher pregnancy and implantation rates
M Cruz et al. compared with the transfer of non-early cleavage embryos (Fenwick et al., 2002; Lonergan et al., 1999; Lundin et al., 2001). Unfortunately, attempts to record the sequential time of cleavage and other relevant events during embryo development to identify embryos of superior quality have proved difficult to implement routinely (Fisch et al., 2001; Neuber et al., 2003). It is well recognized that embryo development is a dynamic process, yet a wealth of information about embryo history has been sacrificed by trying to avoid potentially damaging effects on embryo quality that could be caused by removing embryos from the controlled cultivation temperature and gas environment. Moreover, the impossibility of having an embryologist continuously examining each embryo’s growth and registering these data also points to the need for automated video time-lapse systems; subsequently, the implementation of non-invasive tools for embryo monitoring has been a much needed requirement. This technology is now available and is providing valuable data that can tie the intricate chronological patterns of development and morphological features together with the implantation potential of a specific embryo (Meseguer et al., 2012). Nowadays, intracytoplasmic sperm injection (ICSI) has gained tremendous popularity in assisted reproduction units throughout the world. Despite its initial introduction for severe male factor treatment, it is now utilized in a broader range of cases. In fact, in the USA, 66% of cases are now treated by ICSI (SART, National Data Summary 2010). Due to the intrinsic and different characteristics of the classic IVF and ICSI techniques, the timing of zygote development is influenced by the method of fertilization (Nagy et al., 1994, 1998). During classic IVF, the fertilization takes place in a way similar to natural fertilization but when performing ICSI, several steps of the spermatozoon–oocyte interaction do not occur (Fancsovits et al., 2006), such as sperm penetration through the cumulus cells and the zona pellucida towards the oocyte cytoplasm. The knowledge about the timing of the first cleavage cycle of the human zygote is based on both the conventional IVF (Tesarik and Greco, 1999; Capmany et al., 1996) and the ICSI treatments (Nagy et al., 1994). It has been shown that there can be up to 12 h difference between faster- and slower-developing embryos in the timing of the first cell cycle. However, these differences could be due to the fertilization procedure, as sperm penetration through zona pellucida and oolemma takes a few hours during conventional IVF treatment. Consequently, oocytes fertilized by ICSI undergo pronuclear fading and the first cleavage division around 2–4 h earlier than oocytes derived from classic IVF (Plachot, 2000). These data were recently extended when, by comparing IVF- and ICSI-fertilized oocytes, it was found that ICSI-fertilized 4-cell embryos spent approximately 2.5 h less in the 2-cell stage than IVF 4-cell embryos (Lemmen et al., 2008). Keeping in mind these considerations and biological differences, the selection of a critical ‘starting’ time point, or at least one that is adjusted for insemination technique, is essential so as to facilitate meaningful comparisons of embryo developmental kinetics between the two treatment procedures. There is, however, limited information about the true kinetics of the IVF and ICSI procedures on embryo-quality parameters related to developmental dynamics, and these
Fertilization methods and embryo developmental kinetics factors need to be controlled when aiming to establish the exact timings of optimal embryo developmental events. The aim of this study was to determine whether the IVF or ICSI techniques influence the embryo morphometric and kinetic characteristics and to establish a time point which can be used to compare the two techniques.
Materials and methods This research was conducted at the Instituto Valenciano de Infertilidad IVI Alicante, where a retrospective cohort study analysed 1203 embryos obtained from 178 couples undergoing oocyte donation. The couples received a total of 2379 oocytes (13.4 per recipient, range 7–20); of these, 1637 were normally fertilized and 1203 embryos were fully analysed by time-lapse technology, presenting all the variables that will be described in the following sections. All protocols were approved by an Institutional Review Board (IRB reference: 0711-C-034-MM, October 2008). The project complies with the Spanish law governing assisted reproductive technology (14/2006).
Ovarian stimulation Oocyte donors were healthy woman between 18–35 years old, with regular menstrual cycles, no hereditary or chromosomal diseases, normal karyotype, body mass index 19–29 kg/m2 and negative when screened for sexually transmitted diseases (Garrido et al., 2002). Donors received 1–2 months of treatment with oral contraceptives prior to stimulation for cycle synchronization. Donors underwent a long gonadotrophin-releasing hormone agonist protocol, starting with 0.5 mg leuprolide acetate (Procrin; Abbott, Madrid, Spain) in the mid-luteal phase of the preceding cycle; patients with confirmed adequate pituitary desensitization started their stimulation protocol and the agonist was reduced to 0.25 mg/day until the day of human chorionic gonadotrophin (HCG). Donors were stimulated with 225 IU recombinant FSH (Gonal-F; Merck-Serono, Madrid, Spain) and monitored using ultrasound. HCG (Ovitrelle; Merck-Serono) was administered subcutaneously when at least eight leading follicles had reached a mean diameter 18 mm. Transvaginal retrieval was scheduled 36 h later (Munoz et al., 2012).
Ovum retrieval and IVF and ICSI Recovered oocytes were inseminated using either conventional insemination procedures (IVF) or micromanipulation techniques (ICSI). IVF was usually performed when the sperm sample was of enough quality, although specific criteria were not adopted to decide whether to perform this method of fertilization: sometimes using the embryologist’s judgment and following World Health Organization reference values (total sperm number 39 million per ejaculate and progressive motility 32%; Cooper et al., 2010). Follicles were aspirated and the oocytes were washed in HEPES-buffered medium (Global, Canada) and cultured in Global for Fertilization medium (Global) at 6.0% CO2 and 37.0C for 4 h before oocyte denudation. Oocyte stripping for ICSI was carried out by mechanical pipetting in 1:1 hyaluronidase in Global for Fertilization medium. Likewise, in IVF cycles,
369 inseminated oocytes were also cultured in Global for Fertilization medium until fertilization was confirmed and zygotes were transferred to droplets containing Global medium (Global). Raw sperm concentrations in samples used for ICSI were 49.54 million/ml (95% CI 39.41–59.66) and for IVF averaged 70.86 million/ml (95% CI 62.15–79.57; P = 0.02). The percentage of progressive motility in samples used for ICSI was 35.5 million/ml (95% CI 30.92–40.07) and for IVF was 37.34 million/ml (95% CI 32.96–41.73). Injected or inseminated oocytes were incubated in 20 ll drops of Global for Fertilization medium. Sperm were prepared for IVF in density gradients using All Grad 100 and WASH medium (Global) and the concentration of the insemination drop was fixed at 60,000 motile spermatozoa/ml. Sperm preparation for both IVF and ICSI was carried out in Global for Fertilization medium and ICSI procedures were performed in HEPES-buffered Global for Fertilization medium. Finally, the zygotes were placed inside pre-equilibrated slides (EmbryoSlide; Unisense Fertilitech, Aarhus, Denmark) containing 12 droplets each of 20 ll of Global medium with 1.4 ml overlay of mineral oil (BioCare Europe, Italy) to prevent evaporation. The slides were prepared 24 h in advance and left in the incubator to pre-equilibrate at 6.0% CO2 and 37.0C; after pre-equilibration any bubbles were removed before the zygotes were positioned into individual media droplets on the slide. Fertilization, as indicated by the appearance of 2 pronuclei and 2 polar bodies, was assessed by the EmbryoViewer software from the EmbryoScope (Unisense Fertilitech).
Embryo score and time-lapse annotation Slides containing zygotes were placed in the EmbryoScope immediately after ICSI (day 0) or once fertilization was confirmed (day 1) in IVF cycles. Morphology of cultured embryos was evaluated 48 and 72 h post 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 (Alikani et al., 2000). Blastocysts were scored on day 5 or 6 according to the expansion of blastocoele cavity and integrity of both the inner cell mass and trophoectoderm cells. From the total number of embryos analysed, 82.9% (997/1203) underwent extended culture. The blastocyst rate was 65.2% (650/997) for these embryos. Up to two embryos were transferred on day 3 or 5 after oocyte retrieval; all embryos were selected for transfer (n = 229) in accordance with their conventional morphological scoring parameters on day 3 (n = 107) or according to the blastocyst quality on day 5 (n = 122). The bHCG concentration was determined 13 days after day-3 embryo transfer and 11 days after day-5 transfer. The clinical pregnancy and implantation was confirmed when a gestational sac with fetal heartbeat was visible by ultrasound examination after 7 weeks of pregnancy. By means of a time-lapse system (EmbryoScope), this study determined the timing of a number of development parameters, including the cleavage timings from pronuclear fading (PNF) to 9-cell embryo (t2, t3, t4, t5, t6, t7, t8, t9) as well as time for the formation of morula (M), appearance of blastocoelic cavity (B) and time taken to complete maximal
370 blastocyst expansion (EB). This study also registered some variables related to the duration of the cell cycle: cc2, the second cell cycle, which is the duration of the period as a 2-blastomere embryo (t3–t2); cc3, which is the time taken to pass from a 3-blastomere embryo to a 5-blastomere embryo (t5–t3); s2, synchrony in division from a 2-blastomere embryo to a 4-blastomere embryo (t4–t3); s3, third cell cycle (t4–t3); the length of second embryo cleavage (t4–t2); and finally, the length of the third embryo cleavage (t8–t4). In these recordings, ICSI timing is given relative to the microinjection instant (i.e. the real fertilization) while in classic IVF, the timing is relative to the time of insemination. By integrating morphology and kinetics, an association was established between the patterns and duration of embryo cleavage and the subsequent implantation potential. The optimal timing range for each embryo division was previously described by Meseguer et al. (2011); in this previous study, all cycles were performed by ICSI when the starting point was fixed according the time of microinjection. Optimal embryos with the highest probabilities of implanting are considered to be embryos undergoing development within the timing intervals formerly defined as: t5 = 48.8–56.6 h; s2 0.76 h; and cc2 11.9 h. De-selection criteria for embryos with very low implantation potential have also been described, i.e. those undergoing abrupt division from 1 to 3 or more cells in less than 5 h (CC < 5 h; Rubio et al., 2012). The hierarchical classification procedure related to implantation potential proposed by Meseguer et al. (2011) starts with the binary timing variables t5 and s2. First, if the values of t5 fall inside the optimal range (48.8–56.6 h), the embryo is categorized as A or B; if the value of t5 falls outside the optimal range, the embryo is classified as C or D. If s2 is within the optimal range (0.76 h), the embryo is A or C depending on t5; in a similar way, if s2 >0.76 h, the embryo is B or D, depending also on t5. This classification based on timings was used to compare embryo quality between IVF and ICSI procedures.
Data analysis The developmental parameters were compared using Statistical Package for Social Sciences 19.0 (SPSS, Chicago, IL, USA). Results were analysed using t-test for means and chi-squared test for proportions, considering significance level at P < 0.05. Data are presented with their 95% confidence interval.
Results Fertilization rates for each technique were distributed as follows: for IVF = 71.38% (95% CI 68.80–73.96) and for ICSI = 77.50% (95% CI 74.90–80.10). There were no significant differences between IVF (n = 82) and ICSI (n = 76) as fertilization procedures in relation to clinical pregnancy rates and implantation rates, which were 64.6% (n = 53/82; 95% CI 54.3–75.0%) and 41.0% (n = 57/139; 95% CI 32.8–49.2%), respectively, with IVF, compared with 64.5% (n = 49/76; 95% CI 53.7–75.2%) and 52.3% (n = 68/130; 95% CI 43.7–60.8%) with ICSI.
M Cruz et al.
Embryo kinetics of development and insemination technique The development of ICSI-derived embryos was slightly faster than embryos produced by classical IVF (Table 1), reaching statistical differences in some of the analysed kinetic variables such as: PNF (P = 0.001), which is considered a good prognosis marker of the first cleavage; t2 (P = 0.042), which is related to the time taken to complete first embryo cleavage; t5 (P = 0.016), which provides the best single criterion to select embryos with improved implantation potential; and t9 (P = 0.022), which is the last cell division registered individually before embryo compaction starts. Other events, including M, B and EB, passed within comparable time ranges, none of the minor differences found reaching statistical significance. In order to confirm the suspicion that the observed kinetic differences are due to the intrinsic characteristics of each technique and that they are not reflecting a real delay in IVF embryo development, this work fixed a new starting point (PNF) using an event that could be registered precisely on the time-lapse system, so the hypothetical biases introduced previously could be avoided. Use of this new start point showed no significant differences in embryo kinetics of development between IVF and ICSI (Table 2).
Optimal timing ranges and insemination technique In order to determine whether the insemination method influenced the percentage of embryos in these optimal timings, this work calculated the proportion of embryos inside/outside the following timings defining the inside range as follows: t5 = 48.8–56.6 h; s2 0.76 h; and cc2 11.9 h (Figure 1). Although a trend towards higher values for IVF-derived embryos was observed, the results were not significantly different between the IVF and ICSI groups, so the insemination procedure did not influence embryo quality or implantation potential.
Morphokinetic categories and insemination procedure When the embryo distribution in the algorithm categories was compared, the results were quite similar to those previously obtained (Figure 2; Meseguer et al., 2011). Despite not reaching statistical significance, the proportion of embryos in class D or C was only slightly lower in IVF-derived embryos compared with ICSI-derived embryos. The data followed the same pattern in the rest of the categories, with embryos produced by classic IVF showing slightly better results, although there were significant differences between the two fertilization procedures. A comparison of the proportion of direct-cleavage embryos DC 1–3 (cc2 <5 h) was also performed, with no difference in the proportion of embryos with low implantation potential being found (Figure 3).
Discussion IVF–embryo transfer has been a routinely used procedure in treating infertility for more than 30 years. Its efficacy has
Fertilization methods and embryo developmental kinetics Table 1
371
Embryo kinetics of development according to insemination procedure (t0 = time of fertilization).
Development time from t0 (h)
IVF
ICSI
Total number of embryos PNF t2 t3 t4 t5 t6 t7 t8 t9 cc2 cc3 s2 s3 t4–t2 t8–t4 Day-5 embryos M B EB
622 25.2 (25.0–25.4) 28.5 (27.2–27.8) 39.4 (38.1–40.7) 41.0 (40.5–41.5) 52.1 (51.4–52.8) 54.5 (53.9–55.1) 57.4 (56.6–58.2) 59.4 (58.7–60.1) 62.5 (61.5–63.5) 11.9 (10.7–13.1) 13.4 (12.9–13.7) 2.2 (1.8–2.6) 8.5 (7.8–8.2) 13.5 (13.1–13.9) 19.9 (19.3–20.5) 362 91.0 (90.0–92.0) 103.7 (102.7–104.7) 117.7 (115.7–119.7)
581 23.8 (23.5–24.1) 27.0 (26.7–29.3) 38.2 (37.8–38.6) 40.3 (39.8–40.8) 50.9 (50.1–51.7) 53.7 (53.0–54.4) 56.2 (55.4–57.0) 59.1 (58.3–59.9) 60.7 (59.5–61.9) 11.3 (10.8–11.8) 12.6 (12.1–13.1) 2.1 (1.7–2.5) 9.7 (8.8–10.6) 13.3 (12.9–13.7) 20.2 (19.6–20.8) 288 91.1 (89.9–92.3) 104.9 (103.9–105.9) 117.1 (116.0–119.2)
P-value
0.001 0.042 NS NS 0.016 NS 0.024 NS 0.022 NS NS NS 0.032 NS NS NS NS NS
Values are mean (95% CI). PNF = pronuclear fading; t2 = time to 2-cell embryo; t3 = time to 3-cell embryo; t4 = time to 4-cell embryo; t5 = time to 5-cell embryo; t6 = time to 6-cell embryo; t7 = time to 7-cell embryo; t8 = time to 8-cell embryo; t9 = time to 9-cell embryo; cc2 = t3–t2; cc3 = t5–t3; s2 = t4–t3; s3 = t4–t3; s3 = t8–t5; M = time to morula; B = time to blastocyst; EB = time to expanded blastocyst; NS = not significant.
Table 2 Embryo kinetics of development according to insemination procedure (t0 = pronuclear fading). Development time from t0 (h)
IVF
ICSI
Total embryos t2 t3 t4 t5 t6 t7 t8 t9 Day-5 embryos M B EB
622 3.1 (2.9–3.3) 14.8 (14.2–15.4) 16.8 (16.3–17.3) 28.4 (27.6–29.2) 31.3 (30.5–32.1) 34.0 (33.2–34.8) 36.0 (35.1–36.9) 45.3 (44.3–47.0) 362 66.8 (65.6–67.9) 79.2 (77.9–80.5) 93.7 (91.3–96.1)
581 3.1 (2.8–3.3) 14.1 (13.5–14.7) 16.5 (15.9–17.1) 27.4 (26.5–28.3) 30.5 (29.7–31.3) 33.2 (32.4–34.0) 36.0 (35.1–36.9) 43.7 (42.0–45.4) 288 67.8 (66.5–69.1) 80.3 (79.9–81.7) 94.7 (91.4–98.0)
Values are mean (95% CI). There were no significant differences between the treatments. PNF = pronuclear fading; t2 = time to 2-cell embryo; t3 = time to 3-cell embryo; t4 = time to 4-cell embryo; t5 = time to 5-cell embryo; t6 = time to 6-cell embryo; t7 = time to 7-cell embryo; t8 = time to 8-cell embryo; t9 = time to 9-cell embryo.
considerably increased since the first techniques were used. Some of these newer techniques include more effective stimulation protocols leading to more oocytes and the introduction of micromanipulation techniques, in particular ICSI, making it possible to achieve fertilization in cases of severe
male infertility. Finally, improved embryo culture techniques allow the conversion of oocytes to higher-quality embryos. However, the timing of zygote development can be influenced by the method of fertilization (Nagy et al., 1994; Sakkas et al., 1998) or culture conditions, so it has been suggested that the fertilization procedure influences the length of time elapsed between fertilization and the first cleavage. Sperm penetration through the zona pellucida to the cytoplasm and the presence of immature eggs in the insemination cohort can all impact on the differences between ICSI-derived embryos undergoing pronuclear fading and the first cleavage division compared with IVF-derived embryos (Lemmen et al., 2008). In accordance with other studies (Lemmen et al., 2008; Plachot, 2000), the results of the current study have shown that embryo development after ICSI was significantly faster than after IVF; ICSI-derived embryos had their first cleavage at on average 27.0 h, whereas IVF-fertilized oocytes cleaved at 28.5 h. The observed time difference could simply reflect the time it takes from insemination to fertilization in classic IVF; this time difference could actually be constant but just not significant at the later stages of development due to the larger variability of the late-stage parameters (like morula, blastocyst and expanded blastocyst timing). This time lag exposes the importance of developing a more individualized early scoring cleavage time, or at least one that is adjusted for the insemination technique, before early cleavage scoring between IVF- and ICSI-derived embryos can be compared directly. This need becomes more important when using time-lapse systems where a greater onus is placed on precise timing events.
372
M Cruz et al. 70
66.0 66.4
Embryos (%)
60 50 40
41.2
37.3
34.3
37.2
30 20 10 0
230
199
403 382
t5
256
s2 IVF
216 cc2
ICSI
Figure 1 Optimal timing ranges according to insemination procedure. The following timings defined the inside (optimal) ranges as follows: t5 = 48.8–56.6 h (total IVF 617, ICSI 581); s2 0.76 h (total IVF 611, ICSI 580); and cc2 11.9 h (total IVF 622, ICSI 580). There were no significant differences between the treatments.
45
42.1 42.5
40 Embryos (%)
35 30 25
22.7 23.2
20
20.7 14.3
15 10 5 0
141 135 A
23.2
11.0 64
89 B
IVF
262 247
129 135
C
D
ICSI
Figure 2 Morphokinetic categories according to insemination procedure. A and B = t5 is within optimal range (48.8–56.6 h). C and D = t5 falls outside the optimal range. A and C = s2 is within optimal range (0.76 h), depending on t5. B and D = s2 >0.76 h, depending also on t5 (total IVF = 621; total ICSI = 581). There were no significant differences between the treatments. 100
90.0
90
88.9
Embryos (%)
80 70 60 50 40 30 20 10 0
11.1
10.0 56
502
59
IVF
474 ICSI
DC 1-3
Not DC 1-3
Figure 3 Direct cleavage embryos (DC 1–3) according to insemination procedure (total IVF = 558; total ICSI = 533). There were no significant differences between the treatments.
The large number of confounding factors inherent in clinical embryology must be taken into consideration when analysing embryo development patterns. A potential flaw
in these data could be the changing media in the study, which may introduce subtle differences in energy substrates, phosphate and other micronutrients. Although there are several types of culture media on the market, this work cultured IVF-and ICSI-derived embryos in the same media in order to minimize such factors, thereby reducing the effects of variations introduced by media formulations, which may influence embryo developmental kinetics. However, a recent study (Basile et al., 2013) showed that embryo developmental kinetics are similar for two distinct types of culture media, suggesting that cell kinetics may not be culture media dependent and consequently, that morphokinetic benchmarks can be developed and used irrespective of a laboratory’s choice of media. Another investigation recently reported by Dal Canto et al. (2012) confirmed that the application of standard IVF in contrast to ICSI generates a cleavage delay, maximally present at the 2-cell stage, which is still detectable at the 3-cell stage but completely disappears at the later stages of development. However, the current study was able to establish significant differences in advanced phases of embryo development which conflict with the idea that it
Fertilization methods and embryo developmental kinetics is not necessary to observe IVF- and ICSI-derived embryos at different times and emphasizes that the continuous observations associated with the time-lapse monitoring system are able to identify different cleavage kinetics with developmental significance. As previously suggested and in contrast to ICSI, in classical IVF it is not possible to determine exactly when fertilization occurred so the observed kinetic differences could not be real because the time fixed as the starting point was the same both for IVF and ICSI (i.e. t0 = time of fertilization). In ICSI cycles this value is real, while in IVF it is estimated because of the more variable nature of the insemination process. That is why this study established a new starting point from an event that could be registered precisely in the time-lapse system (i.e. t0 = pronuclear fading). The initial differences disappeared so it can be concluded that the fertilization technique appears to affect embryo cleavage kinetics only because of the specific characteristics of each fertilization method employed. The appearance of a slower embryo development in classic IVF is plausible: sperm penetration through the cumulus cells and the zona pellucida, along with the fusion with the oolemma, occur in approximately 1 hour and these steps are clearly overcome in ICSI cycles. These data justify the selection of a new critical time point so as to maximize the real developmental differences among embryos: the adjusted examination of embryo kinetics of development depending on the insemination procedure gave a more accurate discrimination between these groups. Until the current study, prior reports establishing embryo developmental kinetics between conventional IVF and ICSI have not fixed a new starting point in order to eliminate the variable delay introduced by the fertilization procedure. The results presented here confirm that the delay in IVF-derived embryos is due to the technique itself. Furthermore, some kinetic markers have been developed which are able to predict other important outcomes in embryo development: timing of first cytokinesis in order to predict blastocyst development (Wong et al., 2010); and synchrony in division from 2-blastomere embryos to 4-blastomere embryos, which allows the selection of embryos with a higher implantation potential (Meseguer et al., 2011). These indicators are not influenced by the fertilization starting point and may be then used independently of whether embryos are generated by IVF or ICSI. However, others such as t5 could be affected and then probably modified for IVF cycles. This study group’s intention is to provide an adapted algorithm for IVF embryos, but unfortunately, the frequency of this procedure is low in current practice and it has not been possible to recruit a sufficient number of transferred embryos with known implantation to perform a reliable analysis. The accurate evaluation of embryo implantation becomes crucial when trying to enhance pregnancy outcome and reduce the complication of multiple pregnancies. The morphological appearance and growth rate (cleavage stage) of embryos, which can be observed with non-invasive techniques, are commonly used as parameters to judge embryo quality. The major advantage of performing ICSI is that cases complicated by total fertilization failure can almost be eliminated. However, this application has some inconveniences because it includes higher costs, more technical dif-
373 ficulties and performing an unnecessary invasive procedure in some cases. Moreover, since the ICSI procedure itself is more invasive, some questions regarding safety issues have been raised (Davies et al., 2012). Several studies have demonstrated that there is no increase in the incidence of congenital anomalies in children born after ICSI compared with classic IVF (Bonduelle et al., 1998; Tarlatzis, 1996) while others revealed that infants born after this method might indeed have an excess of major birth defects (Wennerholm et al., 2000). It is possible that the increases in congenital abnormalities observed in some ICSI children are due to factors related to paternal infertility and the use of suboptimal gametes. Another possibility is that the technique itself plays a role; for example, in ICSI oocytes are exposed to hyaluronidase, intense light and fluctuations in temperature and are subject to the creation of an artificial breach in the zona pellucida and oolemma, which can affect embryo quality (Bar-Hava et al., 1997). It has been suggested that some checkpoints are delayed during the ICSI process which could alter the initial cleavage timing difference between IVF and ICSI (Ramalho-Santos et al., 2000). Arguments against classic IVF emphasize a high insemination concentration in cases with moderate and severe teratozoospermia and with an adequate motile sperm fraction (Oehninger et al., 1996), raising the possibility that toxic sperm factors may be released by abnormal and dysfunctional spermatozoa, resulting in damage to the early embryo and leading to a lower proportion of embryos with good morphological scores compared with ICSI. Human spermatozoa can produce reactive oxygen species and it seems that free radical toxicity also contributes to embryonic damage during the culture period necessary for human IVF. Spermatozoa and granulosa cells around the oocytes could also produce metabolic waste, which could cause some adverse effects on oocyte/zygote physiology (Yang et al., 1996). When this work compared embryo quality and implantation potential from a kinetic point of view, the results demonstrated no significant differences between the IVF and ICSI groups, which is agreement with previous studies (Ruiz et al., 1997; Yoeli et al., 2008), although IVF-derived embryos showed a positive trend in the percentages of optimal embryos with a predicted higher implantation potential. Thus, after controlling for maternal effects, embryo morphokinetic characteristics do not seem to be influenced by the mode of fertilization, so it is possible to assume that embryo quality depends on intrinsic factors of the gametes involved rather than on the fertilization process per se. As previously suggested by Sakkas et al., (1998), it would therefore be of interest to ascertain whether the same embryo kinetics are observed repetitively in certain patients, which would indicate that a genetic rather than a technical influence exists. The fact that ICSI-derived embryos cleave significantly faster than IVF-derived embryos did not translate into better embryo quality and these data are in agreement with previous studies (Bonduelle et al., 2002; Dumoulin et al., 2000) which have suggested impaired embryo development in ICSI cycles. Despite IVF-derived embryos starting their initial embryo cleavage later because of the insemination procedure itself, the minimum retardation caused to the oocyte by the delay observed until day 3 of development is somehow corrected. Consequently, when this study evaluated the ‘kinetic qual-
374 ity’ of embryo development, not only did the kinetic differences disappear but also IVF embryos were slightly more numerous in optimal timing ranges with a higher implantation potential, although the difference compared with ICSI embryos was not statistically significant. These results also refute the concept that a high insemination concentration is detrimental during IVF; the attempt to reproduce as real as possible natural fertilization reflects optimal embryo development, which in turns increases the probability of success. Subsequently, the embryo distribution within the algorithm categories as described by Meseguer et al. (2011) followed the same pattern. The integration of morphological and kinetic parameters in order to optimize embryo selection criteria appeared to favour IVF-derived embryos, although this approach failed to reach statistical significance. Interestingly, the proportion of ICSI-derived embryos with DC 1–3 was slightly higher compared with IVF although not significantly (11.1% versus 10.0%). This phenomenon could be explained by the fact that ICSI is usually performed for male factor infertility and so the spermatozoon may be the origin of this slightly poorer morphokinetic characteristic. This fact may also account for why IVF embryos seem better with regard to optimal timing of the embryo kinetics of development and morphology. In conclusion, the intrinsic characteristics of insemination procedures do affect embryo kinetics of development if insemination time is used as the starting point. However, these differences are not reflected in embryo quality and implantation potential. Accordingly, when examining the kinetics of embryo development from IVF- and ICSI-derived embryos, the start time should be reset so as to allow for the different techniques. Further studies are needed with a standardized starting point in order to reduce variability in timings and improve predictive ability.
Presentation This work was presented in part at the 28th annual meeting of the European Society of Human Reproduction and Embryology at Istanbul on 2–4 July 2012.
Acknowledgements The authors acknowledge the assistance and helpful contributions of Dr Dennis Sakkas, Associate Professor, Department of Obstetrics, Gynecology and Reproductive Sciences in Yale University School of Medicine in preparing the manuscript.
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ARTICLE
Oocyte insemination techniques are related to alterations of embryo developmental timing in an oocyte donation model ˜oz a, ´s Garrido a,b, Blanca Gadea a, Manuel Mun Marı´a Cruz a, Nicola ´rez-Cano a, Marcos Meseguer a,b,* Inmaculada Pe a
Instituto Valenciano de Infertilidad, Alicante, Spain;
b
Instituto Universitario IVI Valencia, University of Valencia, Spain
* Corresponding author. E-mail address: [email protected] (M Meseguer). Dr Marcos Meseguer is a senior embryologist, scientific supervisor of Equipo IVI and an associate professor of the Masters in biotechnology from Valencia University. He performed a pre-doctoral fellowship in St Mary’s Hospital, Manchester University, UK. He received his PhD and the European Doctor Degree in obstetrics and gynaecology in 2002 and a Masters in research methods (design and statistics) in 2009. Dr Meseguer has received the prize paper from American Society of Reproductive Medicine, three times the Lalor Foundation International Award from the American Society of Andrology and Reproductive BioMedicine Online Robert Edwards prize paper in 2011. The primary areas of his research are embryology and male infertility.
Abstract Because of the different intrinsic characteristics of the classic IVF and intracytoplasmic sperm injection (ICSI) techniques,
the timing of zygote development can be influenced by the method of fertilization. However, there is no information about the relevance of the insemination procedure on embryo-quality parameters as measured through their developmental dynamics. The aim of this work was to determine if the insemination technique, IVF or ICSI, influences embryo developmental kinetics by examining 1203 embryos from 178 couples undergoing oocyte donation with IVF or ICSI. Using time-lapse information, this work calculated several developmental kinetic variables, from pronuclear fading (PNF) to expanded blastocyst, and also the proportion of optimal embryos in a best time range with a predicted higher implantation potential. Embryo development after ICSI was slightly faster than after IVF; however, when PNF, rather than time of insemination, was established as t0, the differences between the two procedures disappeared. The percentage of optimal embryos showed a trend towards higher values in IVF-derived embryos; however, the difference was not statistically significant. With these results and through the time-lapse monitoring system, it is concluded that it is the fertilization method which determines embryo developmental kinetics if insemination time is used as the starting point. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. KEYWORDS: embryo developmental kinetics, fertilization method, optimal embryos, time lapse
Introduction Little is known about the basic pathways and events of early human preimplantation embryo development, including
factors that would aid in predicting success or failure to develop. Consequently, to increase the chances of pregnancy through IVF, multiple embryos are often transferred to the uterus, despite the concern of well-documented
1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.rbmo.2013.06.017
368 adverse outcomes (Kalra and Barnhart, 2011). Therefore, in order to maximize the pregnancy rate without increasing multiple conceptions, it becomes paramount to develop a method of choosing the embryos that possess the greatest potential to implant. Routinely, embryo morphology and rate of development in culture have been the standard embryo selection techniques in IVF, since the available evidence indicates that a grading system based on these features is a satisfactory indicator of both blastocyst formation (Scott et al., 2000) and implantation potential (Ludwig et al., 2000). While basing the selection of transferable embryos on cell number and quality seems to provide some significant benefit (Cummins et al., 1986), pregnancy rates are still far from the highly theoretical limit of 100%. However, publications from the last decade have shown dramatic improvements compared with the early data and have reported that national success rates are on the rise (Schoolcraft et al., 2010; Shapiro et al., 2011). Furthermore, recently it was demonstrated that the success rate following assisted reproduction increases linearly each year when corrected for maternal age (Cohen et al., 2012). This latter report demonstrates that biological systems altered by technology will consistently improve over time and that this annual increment can be improved by faster incorporation of applied physical or digital systems. For example, the replacement of individual technical expertise with completely automated culture, growth and manipulation systems (Leung et al., 2011) may reduce variation and expedite progress toward a consistent and lasting 100% success rate. It is known that successful implantation, and as a result the pregnancy rate, depends on a competent embryo and a receptive endometrium with a synchronized dialogue between the maternal and embryonic tissues. However, pregnancy rates following IVF attempts remain low, where two out of three IVF cycles fail to result in a pregnancy and more than eight out of ten transferred embryos fail to implant (Kovalevsky and Patrizio, 2005). Given that many embryos, catalogued as displaying good prognosis using classical morphological assessment protocols, fail to implant, while other low-rated embryos are sometimes able to initiate and maintain a pregnancy, it is clear that the discriminative power of this analysis can be improved (Seli et al., 2010). A recognized shortcoming of the current methods of embryo evaluation is that they are static and only obtain information from very short intervals of time through embryo development, thus limiting a true assessment of the intricacies of morphological characteristics and cell number. McKiernan and Bavister (Bavister, 1995) highlighted this problem by stating that the examination of embryos at arbitrary time points during embryo development can be quite misleading with respect to categorizing the stage of development reached and ‘timelines’ of development. The importance of establishing the precise timing of development has been recognized in animal models, where embryos reaching the 8-cell stage in a defined time frame presented a greater ability to reach the blastocyst stage and a higher viability when transferred (Sakkas et al., 1998). Even in the human, the selection and transfer of early cleavage 2-cell embryos has consistently been shown to provide higher pregnancy and implantation rates
M Cruz et al. compared with the transfer of non-early cleavage embryos (Fenwick et al., 2002; Lonergan et al., 1999; Lundin et al., 2001). Unfortunately, attempts to record the sequential time of cleavage and other relevant events during embryo development to identify embryos of superior quality have proved difficult to implement routinely (Fisch et al., 2001; Neuber et al., 2003). It is well recognized that embryo development is a dynamic process, yet a wealth of information about embryo history has been sacrificed by trying to avoid potentially damaging effects on embryo quality that could be caused by removing embryos from the controlled cultivation temperature and gas environment. Moreover, the impossibility of having an embryologist continuously examining each embryo’s growth and registering these data also points to the need for automated video time-lapse systems; subsequently, the implementation of non-invasive tools for embryo monitoring has been a much needed requirement. This technology is now available and is providing valuable data that can tie the intricate chronological patterns of development and morphological features together with the implantation potential of a specific embryo (Meseguer et al., 2012). Nowadays, intracytoplasmic sperm injection (ICSI) has gained tremendous popularity in assisted reproduction units throughout the world. Despite its initial introduction for severe male factor treatment, it is now utilized in a broader range of cases. In fact, in the USA, 66% of cases are now treated by ICSI (SART, National Data Summary 2010). Due to the intrinsic and different characteristics of the classic IVF and ICSI techniques, the timing of zygote development is influenced by the method of fertilization (Nagy et al., 1994, 1998). During classic IVF, the fertilization takes place in a way similar to natural fertilization but when performing ICSI, several steps of the spermatozoon–oocyte interaction do not occur (Fancsovits et al., 2006), such as sperm penetration through the cumulus cells and the zona pellucida towards the oocyte cytoplasm. The knowledge about the timing of the first cleavage cycle of the human zygote is based on both the conventional IVF (Tesarik and Greco, 1999; Capmany et al., 1996) and the ICSI treatments (Nagy et al., 1994). It has been shown that there can be up to 12 h difference between faster- and slower-developing embryos in the timing of the first cell cycle. However, these differences could be due to the fertilization procedure, as sperm penetration through zona pellucida and oolemma takes a few hours during conventional IVF treatment. Consequently, oocytes fertilized by ICSI undergo pronuclear fading and the first cleavage division around 2–4 h earlier than oocytes derived from classic IVF (Plachot, 2000). These data were recently extended when, by comparing IVF- and ICSI-fertilized oocytes, it was found that ICSI-fertilized 4-cell embryos spent approximately 2.5 h less in the 2-cell stage than IVF 4-cell embryos (Lemmen et al., 2008). Keeping in mind these considerations and biological differences, the selection of a critical ‘starting’ time point, or at least one that is adjusted for insemination technique, is essential so as to facilitate meaningful comparisons of embryo developmental kinetics between the two treatment procedures. There is, however, limited information about the true kinetics of the IVF and ICSI procedures on embryo-quality parameters related to developmental dynamics, and these
Fertilization methods and embryo developmental kinetics factors need to be controlled when aiming to establish the exact timings of optimal embryo developmental events. The aim of this study was to determine whether the IVF or ICSI techniques influence the embryo morphometric and kinetic characteristics and to establish a time point which can be used to compare the two techniques.
Materials and methods This research was conducted at the Instituto Valenciano de Infertilidad IVI Alicante, where a retrospective cohort study analysed 1203 embryos obtained from 178 couples undergoing oocyte donation. The couples received a total of 2379 oocytes (13.4 per recipient, range 7–20); of these, 1637 were normally fertilized and 1203 embryos were fully analysed by time-lapse technology, presenting all the variables that will be described in the following sections. All protocols were approved by an Institutional Review Board (IRB reference: 0711-C-034-MM, October 2008). The project complies with the Spanish law governing assisted reproductive technology (14/2006).
Ovarian stimulation Oocyte donors were healthy woman between 18–35 years old, with regular menstrual cycles, no hereditary or chromosomal diseases, normal karyotype, body mass index 19–29 kg/m2 and negative when screened for sexually transmitted diseases (Garrido et al., 2002). Donors received 1–2 months of treatment with oral contraceptives prior to stimulation for cycle synchronization. Donors underwent a long gonadotrophin-releasing hormone agonist protocol, starting with 0.5 mg leuprolide acetate (Procrin; Abbott, Madrid, Spain) in the mid-luteal phase of the preceding cycle; patients with confirmed adequate pituitary desensitization started their stimulation protocol and the agonist was reduced to 0.25 mg/day until the day of human chorionic gonadotrophin (HCG). Donors were stimulated with 225 IU recombinant FSH (Gonal-F; Merck-Serono, Madrid, Spain) and monitored using ultrasound. HCG (Ovitrelle; Merck-Serono) was administered subcutaneously when at least eight leading follicles had reached a mean diameter 18 mm. Transvaginal retrieval was scheduled 36 h later (Munoz et al., 2012).
Ovum retrieval and IVF and ICSI Recovered oocytes were inseminated using either conventional insemination procedures (IVF) or micromanipulation techniques (ICSI). IVF was usually performed when the sperm sample was of enough quality, although specific criteria were not adopted to decide whether to perform this method of fertilization: sometimes using the embryologist’s judgment and following World Health Organization reference values (total sperm number 39 million per ejaculate and progressive motility 32%; Cooper et al., 2010). Follicles were aspirated and the oocytes were washed in HEPES-buffered medium (Global, Canada) and cultured in Global for Fertilization medium (Global) at 6.0% CO2 and 37.0C for 4 h before oocyte denudation. Oocyte stripping for ICSI was carried out by mechanical pipetting in 1:1 hyaluronidase in Global for Fertilization medium. Likewise, in IVF cycles,
369 inseminated oocytes were also cultured in Global for Fertilization medium until fertilization was confirmed and zygotes were transferred to droplets containing Global medium (Global). Raw sperm concentrations in samples used for ICSI were 49.54 million/ml (95% CI 39.41–59.66) and for IVF averaged 70.86 million/ml (95% CI 62.15–79.57; P = 0.02). The percentage of progressive motility in samples used for ICSI was 35.5 million/ml (95% CI 30.92–40.07) and for IVF was 37.34 million/ml (95% CI 32.96–41.73). Injected or inseminated oocytes were incubated in 20 ll drops of Global for Fertilization medium. Sperm were prepared for IVF in density gradients using All Grad 100 and WASH medium (Global) and the concentration of the insemination drop was fixed at 60,000 motile spermatozoa/ml. Sperm preparation for both IVF and ICSI was carried out in Global for Fertilization medium and ICSI procedures were performed in HEPES-buffered Global for Fertilization medium. Finally, the zygotes were placed inside pre-equilibrated slides (EmbryoSlide; Unisense Fertilitech, Aarhus, Denmark) containing 12 droplets each of 20 ll of Global medium with 1.4 ml overlay of mineral oil (BioCare Europe, Italy) to prevent evaporation. The slides were prepared 24 h in advance and left in the incubator to pre-equilibrate at 6.0% CO2 and 37.0C; after pre-equilibration any bubbles were removed before the zygotes were positioned into individual media droplets on the slide. Fertilization, as indicated by the appearance of 2 pronuclei and 2 polar bodies, was assessed by the EmbryoViewer software from the EmbryoScope (Unisense Fertilitech).
Embryo score and time-lapse annotation Slides containing zygotes were placed in the EmbryoScope immediately after ICSI (day 0) or once fertilization was confirmed (day 1) in IVF cycles. Morphology of cultured embryos was evaluated 48 and 72 h post 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 (Alikani et al., 2000). Blastocysts were scored on day 5 or 6 according to the expansion of blastocoele cavity and integrity of both the inner cell mass and trophoectoderm cells. From the total number of embryos analysed, 82.9% (997/1203) underwent extended culture. The blastocyst rate was 65.2% (650/997) for these embryos. Up to two embryos were transferred on day 3 or 5 after oocyte retrieval; all embryos were selected for transfer (n = 229) in accordance with their conventional morphological scoring parameters on day 3 (n = 107) or according to the blastocyst quality on day 5 (n = 122). The bHCG concentration was determined 13 days after day-3 embryo transfer and 11 days after day-5 transfer. The clinical pregnancy and implantation was confirmed when a gestational sac with fetal heartbeat was visible by ultrasound examination after 7 weeks of pregnancy. By means of a time-lapse system (EmbryoScope), this study determined the timing of a number of development parameters, including the cleavage timings from pronuclear fading (PNF) to 9-cell embryo (t2, t3, t4, t5, t6, t7, t8, t9) as well as time for the formation of morula (M), appearance of blastocoelic cavity (B) and time taken to complete maximal
370 blastocyst expansion (EB). This study also registered some variables related to the duration of the cell cycle: cc2, the second cell cycle, which is the duration of the period as a 2-blastomere embryo (t3–t2); cc3, which is the time taken to pass from a 3-blastomere embryo to a 5-blastomere embryo (t5–t3); s2, synchrony in division from a 2-blastomere embryo to a 4-blastomere embryo (t4–t3); s3, third cell cycle (t4–t3); the length of second embryo cleavage (t4–t2); and finally, the length of the third embryo cleavage (t8–t4). In these recordings, ICSI timing is given relative to the microinjection instant (i.e. the real fertilization) while in classic IVF, the timing is relative to the time of insemination. By integrating morphology and kinetics, an association was established between the patterns and duration of embryo cleavage and the subsequent implantation potential. The optimal timing range for each embryo division was previously described by Meseguer et al. (2011); in this previous study, all cycles were performed by ICSI when the starting point was fixed according the time of microinjection. Optimal embryos with the highest probabilities of implanting are considered to be embryos undergoing development within the timing intervals formerly defined as: t5 = 48.8–56.6 h; s2 0.76 h; and cc2 11.9 h. De-selection criteria for embryos with very low implantation potential have also been described, i.e. those undergoing abrupt division from 1 to 3 or more cells in less than 5 h (CC < 5 h; Rubio et al., 2012). The hierarchical classification procedure related to implantation potential proposed by Meseguer et al. (2011) starts with the binary timing variables t5 and s2. First, if the values of t5 fall inside the optimal range (48.8–56.6 h), the embryo is categorized as A or B; if the value of t5 falls outside the optimal range, the embryo is classified as C or D. If s2 is within the optimal range (0.76 h), the embryo is A or C depending on t5; in a similar way, if s2 >0.76 h, the embryo is B or D, depending also on t5. This classification based on timings was used to compare embryo quality between IVF and ICSI procedures.
Data analysis The developmental parameters were compared using Statistical Package for Social Sciences 19.0 (SPSS, Chicago, IL, USA). Results were analysed using t-test for means and chi-squared test for proportions, considering significance level at P < 0.05. Data are presented with their 95% confidence interval.
Results Fertilization rates for each technique were distributed as follows: for IVF = 71.38% (95% CI 68.80–73.96) and for ICSI = 77.50% (95% CI 74.90–80.10). There were no significant differences between IVF (n = 82) and ICSI (n = 76) as fertilization procedures in relation to clinical pregnancy rates and implantation rates, which were 64.6% (n = 53/82; 95% CI 54.3–75.0%) and 41.0% (n = 57/139; 95% CI 32.8–49.2%), respectively, with IVF, compared with 64.5% (n = 49/76; 95% CI 53.7–75.2%) and 52.3% (n = 68/130; 95% CI 43.7–60.8%) with ICSI.
M Cruz et al.
Embryo kinetics of development and insemination technique The development of ICSI-derived embryos was slightly faster than embryos produced by classical IVF (Table 1), reaching statistical differences in some of the analysed kinetic variables such as: PNF (P = 0.001), which is considered a good prognosis marker of the first cleavage; t2 (P = 0.042), which is related to the time taken to complete first embryo cleavage; t5 (P = 0.016), which provides the best single criterion to select embryos with improved implantation potential; and t9 (P = 0.022), which is the last cell division registered individually before embryo compaction starts. Other events, including M, B and EB, passed within comparable time ranges, none of the minor differences found reaching statistical significance. In order to confirm the suspicion that the observed kinetic differences are due to the intrinsic characteristics of each technique and that they are not reflecting a real delay in IVF embryo development, this work fixed a new starting point (PNF) using an event that could be registered precisely on the time-lapse system, so the hypothetical biases introduced previously could be avoided. Use of this new start point showed no significant differences in embryo kinetics of development between IVF and ICSI (Table 2).
Optimal timing ranges and insemination technique In order to determine whether the insemination method influenced the percentage of embryos in these optimal timings, this work calculated the proportion of embryos inside/outside the following timings defining the inside range as follows: t5 = 48.8–56.6 h; s2 0.76 h; and cc2 11.9 h (Figure 1). Although a trend towards higher values for IVF-derived embryos was observed, the results were not significantly different between the IVF and ICSI groups, so the insemination procedure did not influence embryo quality or implantation potential.
Morphokinetic categories and insemination procedure When the embryo distribution in the algorithm categories was compared, the results were quite similar to those previously obtained (Figure 2; Meseguer et al., 2011). Despite not reaching statistical significance, the proportion of embryos in class D or C was only slightly lower in IVF-derived embryos compared with ICSI-derived embryos. The data followed the same pattern in the rest of the categories, with embryos produced by classic IVF showing slightly better results, although there were significant differences between the two fertilization procedures. A comparison of the proportion of direct-cleavage embryos DC 1–3 (cc2 <5 h) was also performed, with no difference in the proportion of embryos with low implantation potential being found (Figure 3).
Discussion IVF–embryo transfer has been a routinely used procedure in treating infertility for more than 30 years. Its efficacy has
Fertilization methods and embryo developmental kinetics Table 1
371
Embryo kinetics of development according to insemination procedure (t0 = time of fertilization).
Development time from t0 (h)
IVF
ICSI
Total number of embryos PNF t2 t3 t4 t5 t6 t7 t8 t9 cc2 cc3 s2 s3 t4–t2 t8–t4 Day-5 embryos M B EB
622 25.2 (25.0–25.4) 28.5 (27.2–27.8) 39.4 (38.1–40.7) 41.0 (40.5–41.5) 52.1 (51.4–52.8) 54.5 (53.9–55.1) 57.4 (56.6–58.2) 59.4 (58.7–60.1) 62.5 (61.5–63.5) 11.9 (10.7–13.1) 13.4 (12.9–13.7) 2.2 (1.8–2.6) 8.5 (7.8–8.2) 13.5 (13.1–13.9) 19.9 (19.3–20.5) 362 91.0 (90.0–92.0) 103.7 (102.7–104.7) 117.7 (115.7–119.7)
581 23.8 (23.5–24.1) 27.0 (26.7–29.3) 38.2 (37.8–38.6) 40.3 (39.8–40.8) 50.9 (50.1–51.7) 53.7 (53.0–54.4) 56.2 (55.4–57.0) 59.1 (58.3–59.9) 60.7 (59.5–61.9) 11.3 (10.8–11.8) 12.6 (12.1–13.1) 2.1 (1.7–2.5) 9.7 (8.8–10.6) 13.3 (12.9–13.7) 20.2 (19.6–20.8) 288 91.1 (89.9–92.3) 104.9 (103.9–105.9) 117.1 (116.0–119.2)
P-value
0.001 0.042 NS NS 0.016 NS 0.024 NS 0.022 NS NS NS 0.032 NS NS NS NS NS
Values are mean (95% CI). PNF = pronuclear fading; t2 = time to 2-cell embryo; t3 = time to 3-cell embryo; t4 = time to 4-cell embryo; t5 = time to 5-cell embryo; t6 = time to 6-cell embryo; t7 = time to 7-cell embryo; t8 = time to 8-cell embryo; t9 = time to 9-cell embryo; cc2 = t3–t2; cc3 = t5–t3; s2 = t4–t3; s3 = t4–t3; s3 = t8–t5; M = time to morula; B = time to blastocyst; EB = time to expanded blastocyst; NS = not significant.
Table 2 Embryo kinetics of development according to insemination procedure (t0 = pronuclear fading). Development time from t0 (h)
IVF
ICSI
Total embryos t2 t3 t4 t5 t6 t7 t8 t9 Day-5 embryos M B EB
622 3.1 (2.9–3.3) 14.8 (14.2–15.4) 16.8 (16.3–17.3) 28.4 (27.6–29.2) 31.3 (30.5–32.1) 34.0 (33.2–34.8) 36.0 (35.1–36.9) 45.3 (44.3–47.0) 362 66.8 (65.6–67.9) 79.2 (77.9–80.5) 93.7 (91.3–96.1)
581 3.1 (2.8–3.3) 14.1 (13.5–14.7) 16.5 (15.9–17.1) 27.4 (26.5–28.3) 30.5 (29.7–31.3) 33.2 (32.4–34.0) 36.0 (35.1–36.9) 43.7 (42.0–45.4) 288 67.8 (66.5–69.1) 80.3 (79.9–81.7) 94.7 (91.4–98.0)
Values are mean (95% CI). There were no significant differences between the treatments. PNF = pronuclear fading; t2 = time to 2-cell embryo; t3 = time to 3-cell embryo; t4 = time to 4-cell embryo; t5 = time to 5-cell embryo; t6 = time to 6-cell embryo; t7 = time to 7-cell embryo; t8 = time to 8-cell embryo; t9 = time to 9-cell embryo.
considerably increased since the first techniques were used. Some of these newer techniques include more effective stimulation protocols leading to more oocytes and the introduction of micromanipulation techniques, in particular ICSI, making it possible to achieve fertilization in cases of severe
male infertility. Finally, improved embryo culture techniques allow the conversion of oocytes to higher-quality embryos. However, the timing of zygote development can be influenced by the method of fertilization (Nagy et al., 1994; Sakkas et al., 1998) or culture conditions, so it has been suggested that the fertilization procedure influences the length of time elapsed between fertilization and the first cleavage. Sperm penetration through the zona pellucida to the cytoplasm and the presence of immature eggs in the insemination cohort can all impact on the differences between ICSI-derived embryos undergoing pronuclear fading and the first cleavage division compared with IVF-derived embryos (Lemmen et al., 2008). In accordance with other studies (Lemmen et al., 2008; Plachot, 2000), the results of the current study have shown that embryo development after ICSI was significantly faster than after IVF; ICSI-derived embryos had their first cleavage at on average 27.0 h, whereas IVF-fertilized oocytes cleaved at 28.5 h. The observed time difference could simply reflect the time it takes from insemination to fertilization in classic IVF; this time difference could actually be constant but just not significant at the later stages of development due to the larger variability of the late-stage parameters (like morula, blastocyst and expanded blastocyst timing). This time lag exposes the importance of developing a more individualized early scoring cleavage time, or at least one that is adjusted for the insemination technique, before early cleavage scoring between IVF- and ICSI-derived embryos can be compared directly. This need becomes more important when using time-lapse systems where a greater onus is placed on precise timing events.
372
M Cruz et al. 70
66.0 66.4
Embryos (%)
60 50 40
41.2
37.3
34.3
37.2
30 20 10 0
230
199
403 382
t5
256
s2 IVF
216 cc2
ICSI
Figure 1 Optimal timing ranges according to insemination procedure. The following timings defined the inside (optimal) ranges as follows: t5 = 48.8–56.6 h (total IVF 617, ICSI 581); s2 0.76 h (total IVF 611, ICSI 580); and cc2 11.9 h (total IVF 622, ICSI 580). There were no significant differences between the treatments.
45
42.1 42.5
40 Embryos (%)
35 30 25
22.7 23.2
20
20.7 14.3
15 10 5 0
141 135 A
23.2
11.0 64
89 B
IVF
262 247
129 135
C
D
ICSI
Figure 2 Morphokinetic categories according to insemination procedure. A and B = t5 is within optimal range (48.8–56.6 h). C and D = t5 falls outside the optimal range. A and C = s2 is within optimal range (0.76 h), depending on t5. B and D = s2 >0.76 h, depending also on t5 (total IVF = 621; total ICSI = 581). There were no significant differences between the treatments. 100
90.0
90
88.9
Embryos (%)
80 70 60 50 40 30 20 10 0
11.1
10.0 56
502
59
IVF
474 ICSI
DC 1-3
Not DC 1-3
Figure 3 Direct cleavage embryos (DC 1–3) according to insemination procedure (total IVF = 558; total ICSI = 533). There were no significant differences between the treatments.
The large number of confounding factors inherent in clinical embryology must be taken into consideration when analysing embryo development patterns. A potential flaw
in these data could be the changing media in the study, which may introduce subtle differences in energy substrates, phosphate and other micronutrients. Although there are several types of culture media on the market, this work cultured IVF-and ICSI-derived embryos in the same media in order to minimize such factors, thereby reducing the effects of variations introduced by media formulations, which may influence embryo developmental kinetics. However, a recent study (Basile et al., 2013) showed that embryo developmental kinetics are similar for two distinct types of culture media, suggesting that cell kinetics may not be culture media dependent and consequently, that morphokinetic benchmarks can be developed and used irrespective of a laboratory’s choice of media. Another investigation recently reported by Dal Canto et al. (2012) confirmed that the application of standard IVF in contrast to ICSI generates a cleavage delay, maximally present at the 2-cell stage, which is still detectable at the 3-cell stage but completely disappears at the later stages of development. However, the current study was able to establish significant differences in advanced phases of embryo development which conflict with the idea that it
Fertilization methods and embryo developmental kinetics is not necessary to observe IVF- and ICSI-derived embryos at different times and emphasizes that the continuous observations associated with the time-lapse monitoring system are able to identify different cleavage kinetics with developmental significance. As previously suggested and in contrast to ICSI, in classical IVF it is not possible to determine exactly when fertilization occurred so the observed kinetic differences could not be real because the time fixed as the starting point was the same both for IVF and ICSI (i.e. t0 = time of fertilization). In ICSI cycles this value is real, while in IVF it is estimated because of the more variable nature of the insemination process. That is why this study established a new starting point from an event that could be registered precisely in the time-lapse system (i.e. t0 = pronuclear fading). The initial differences disappeared so it can be concluded that the fertilization technique appears to affect embryo cleavage kinetics only because of the specific characteristics of each fertilization method employed. The appearance of a slower embryo development in classic IVF is plausible: sperm penetration through the cumulus cells and the zona pellucida, along with the fusion with the oolemma, occur in approximately 1 hour and these steps are clearly overcome in ICSI cycles. These data justify the selection of a new critical time point so as to maximize the real developmental differences among embryos: the adjusted examination of embryo kinetics of development depending on the insemination procedure gave a more accurate discrimination between these groups. Until the current study, prior reports establishing embryo developmental kinetics between conventional IVF and ICSI have not fixed a new starting point in order to eliminate the variable delay introduced by the fertilization procedure. The results presented here confirm that the delay in IVF-derived embryos is due to the technique itself. Furthermore, some kinetic markers have been developed which are able to predict other important outcomes in embryo development: timing of first cytokinesis in order to predict blastocyst development (Wong et al., 2010); and synchrony in division from 2-blastomere embryos to 4-blastomere embryos, which allows the selection of embryos with a higher implantation potential (Meseguer et al., 2011). These indicators are not influenced by the fertilization starting point and may be then used independently of whether embryos are generated by IVF or ICSI. However, others such as t5 could be affected and then probably modified for IVF cycles. This study group’s intention is to provide an adapted algorithm for IVF embryos, but unfortunately, the frequency of this procedure is low in current practice and it has not been possible to recruit a sufficient number of transferred embryos with known implantation to perform a reliable analysis. The accurate evaluation of embryo implantation becomes crucial when trying to enhance pregnancy outcome and reduce the complication of multiple pregnancies. The morphological appearance and growth rate (cleavage stage) of embryos, which can be observed with non-invasive techniques, are commonly used as parameters to judge embryo quality. The major advantage of performing ICSI is that cases complicated by total fertilization failure can almost be eliminated. However, this application has some inconveniences because it includes higher costs, more technical dif-
373 ficulties and performing an unnecessary invasive procedure in some cases. Moreover, since the ICSI procedure itself is more invasive, some questions regarding safety issues have been raised (Davies et al., 2012). Several studies have demonstrated that there is no increase in the incidence of congenital anomalies in children born after ICSI compared with classic IVF (Bonduelle et al., 1998; Tarlatzis, 1996) while others revealed that infants born after this method might indeed have an excess of major birth defects (Wennerholm et al., 2000). It is possible that the increases in congenital abnormalities observed in some ICSI children are due to factors related to paternal infertility and the use of suboptimal gametes. Another possibility is that the technique itself plays a role; for example, in ICSI oocytes are exposed to hyaluronidase, intense light and fluctuations in temperature and are subject to the creation of an artificial breach in the zona pellucida and oolemma, which can affect embryo quality (Bar-Hava et al., 1997). It has been suggested that some checkpoints are delayed during the ICSI process which could alter the initial cleavage timing difference between IVF and ICSI (Ramalho-Santos et al., 2000). Arguments against classic IVF emphasize a high insemination concentration in cases with moderate and severe teratozoospermia and with an adequate motile sperm fraction (Oehninger et al., 1996), raising the possibility that toxic sperm factors may be released by abnormal and dysfunctional spermatozoa, resulting in damage to the early embryo and leading to a lower proportion of embryos with good morphological scores compared with ICSI. Human spermatozoa can produce reactive oxygen species and it seems that free radical toxicity also contributes to embryonic damage during the culture period necessary for human IVF. Spermatozoa and granulosa cells around the oocytes could also produce metabolic waste, which could cause some adverse effects on oocyte/zygote physiology (Yang et al., 1996). When this work compared embryo quality and implantation potential from a kinetic point of view, the results demonstrated no significant differences between the IVF and ICSI groups, which is agreement with previous studies (Ruiz et al., 1997; Yoeli et al., 2008), although IVF-derived embryos showed a positive trend in the percentages of optimal embryos with a predicted higher implantation potential. Thus, after controlling for maternal effects, embryo morphokinetic characteristics do not seem to be influenced by the mode of fertilization, so it is possible to assume that embryo quality depends on intrinsic factors of the gametes involved rather than on the fertilization process per se. As previously suggested by Sakkas et al., (1998), it would therefore be of interest to ascertain whether the same embryo kinetics are observed repetitively in certain patients, which would indicate that a genetic rather than a technical influence exists. The fact that ICSI-derived embryos cleave significantly faster than IVF-derived embryos did not translate into better embryo quality and these data are in agreement with previous studies (Bonduelle et al., 2002; Dumoulin et al., 2000) which have suggested impaired embryo development in ICSI cycles. Despite IVF-derived embryos starting their initial embryo cleavage later because of the insemination procedure itself, the minimum retardation caused to the oocyte by the delay observed until day 3 of development is somehow corrected. Consequently, when this study evaluated the ‘kinetic qual-
374 ity’ of embryo development, not only did the kinetic differences disappear but also IVF embryos were slightly more numerous in optimal timing ranges with a higher implantation potential, although the difference compared with ICSI embryos was not statistically significant. These results also refute the concept that a high insemination concentration is detrimental during IVF; the attempt to reproduce as real as possible natural fertilization reflects optimal embryo development, which in turns increases the probability of success. Subsequently, the embryo distribution within the algorithm categories as described by Meseguer et al. (2011) followed the same pattern. The integration of morphological and kinetic parameters in order to optimize embryo selection criteria appeared to favour IVF-derived embryos, although this approach failed to reach statistical significance. Interestingly, the proportion of ICSI-derived embryos with DC 1–3 was slightly higher compared with IVF although not significantly (11.1% versus 10.0%). This phenomenon could be explained by the fact that ICSI is usually performed for male factor infertility and so the spermatozoon may be the origin of this slightly poorer morphokinetic characteristic. This fact may also account for why IVF embryos seem better with regard to optimal timing of the embryo kinetics of development and morphology. In conclusion, the intrinsic characteristics of insemination procedures do affect embryo kinetics of development if insemination time is used as the starting point. However, these differences are not reflected in embryo quality and implantation potential. Accordingly, when examining the kinetics of embryo development from IVF- and ICSI-derived embryos, the start time should be reset so as to allow for the different techniques. Further studies are needed with a standardized starting point in order to reduce variability in timings and improve predictive ability.
Presentation This work was presented in part at the 28th annual meeting of the European Society of Human Reproduction and Embryology at Istanbul on 2–4 July 2012.
Acknowledgements The authors acknowledge the assistance and helpful contributions of Dr Dennis Sakkas, Associate Professor, Department of Obstetrics, Gynecology and Reproductive Sciences in Yale University School of Medicine in preparing the manuscript.
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