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Influence of oocyte dysmorphisms on blastocyst formation and quality
Influence of oocyte dysmorphisms on blastocyst formation and quality Daniela Paes Almeida Ferreira Braga, D.V.M., M.Sc.,a,b Amanda S. Setti, M.Sc.,a,b ssia S. Figueira, M.Sc.,a Roge rio Bonassi Machado, M.D., Ph.D.,c Assumpto Iaconelli Jr., M.D.,a Rita de Ca a,b and Edson Borges Jr., M.D., Ph.D. a ~o Assistida, Sa ~o Paulo, Sa ~ o Paulo, Brazil; b Instituto Sapientiae, Centro de Estudos e Pesquisa Fertility, Centro de Fertilizac¸a ~ o Humana Assistida, Sa ~ o Paulo, Sa ~o Paulo, Brazil; and c Faculdade de Medicina de Jundiaí, Jundiaí, em Reproduc¸a ~o Paulo, Brazil Sa
Objective: To identify the association between oocyte dysmorphisms and blastocyst developmental competence. Design: Case–control study. Setting: Private assisted reproduction center. Patient(s): This study included 5,516 embryos, which were obtained from 605 patients undergoing intracytoplasmic sperm injection cycles in which ET was performed on day 5 of embryo development. Intervention(s): Intracytoplasmic sperm injection and ET. Main Outcome Measure(s): The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm cells, were correlated with oocyte dysmorphisms. Result(s): Of the 5,516 embryos evaluated, 2,920 (52.9%) reached the blastocyst stage. The blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth endoplasmic reticulum clusters, large perivitelline space, and shape abnormalities. The presence of a large perivitelline space also decreased the ICM and trophectoderm cell quality. Aggregates of smooth endoplasmic reticulum clusters also decreased the ICM quality, pregnancy rate, and the miscarriage rate. Conclusion(s): The results presented here suggest that the individual identification of oocyte dymorphisms may be a prognostic tool for blastocyst development and quality. However, the results do not diminish the importance of cleavage-stage embryo morphology assessment to predict blastocyst development competence. (Fertil SterilÒ 2013;100:748–54. Use your smartphone Ó2013 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Oocyte, blastocyst, perivitelline space, smooth endoplasmic reticulum, oocyte and connect to the shape Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/bragadpdaf-oocyte-dysmorphisms-blastocyst-formation/
O
ocyte quality has been regarded as a variable that influences the implantation potential of derived embryos. To date there have been many published reports on the impact of oocyte morphology on embryo development (1–4). The most frequently observed oocyte morphologic variations are cytoplasmic, including changes in color, granularity, and homogeneity, as well as cytoplasmic incorporations.
Extracytoplasmic variations are deviations from normal perivitelline space (PVS), zona pellucida (ZP) color, and oocyte shape (5). However, the predictive value of the criteria used in these previous studies is still controversial (2, 6–19). An extended embryo culture and the transfer of blastocyst-stage embryos are associated with increased implantation rates compared with the transfer of cleavage-stage embryos
Received March 1, 2013; revised May 9, 2013; accepted May 15, 2013; published online June 12, 2013. D.P.A.F.B. has nothing to disclose. A.S.S. has nothing to disclose. R.d.C.S.F. has nothing to disclose. R.B.M. has nothing to disclose. A.I. has nothing to disclose. E.B. has nothing to disclose. ~o Assistida, Av. BrigadeReprint requests: Edson Borges Jr., M.D., Ph.D., Fertility, Centro de Fertilizac¸a ^ nio 4545, 01401-002 Sa ~o Paulo, Sa ~o Paulo, Brazil (E-mail: [email protected]). iro Luis Anto Fertility and Sterility® Vol. 100, No. 3, September 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.05.021 748
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(20, 21). A prolonged culture period allows for a better selection of more-advanced embryos that are not arrested because the laboratory assessment is undertaken after the embryonic genome has begun to be expressed (22). In addition, it has been reported that better synchronization between endometrium function and embryo development is possible with blastocyst-stage ETs (23, 24). Another potential benefit of blastocyst culture would be that at day 5, uterine contractility is decreased as compared with days 2 or 3 (25). However, the proportion of embryos that reach the blastocyst stage is still unpredictable (26–28), and some assisted reproduction centers are reluctant to adopt extended embryo VOL. 100 NO. 3 / SEPTEMBER 2013
Fertility and Sterility® cultures to avoid any ET cancellation due to an unpredictable rate of blastocyst development. Various factors may be responsible for this variation in the rate of blastocyst formation. Considering the vital role played by the oocyte in the developmental process, the noninvasive identification of oocyte dysmorphisms before fertilization would be extremely useful in predicting the rate of blastocyst formation. There is growing evidence in animal studies to support the idea that recurrent failure in assisted reproduction could be explained by low oocyte quality (29–32). However, although animal studies seem to support an association between oocyte and blastocyst quality, human studies are scarce. Therefore, the goal of the present study was to identify the association between oocyte dysmorphisms and blastocyst developmental competence.
MATERIALS AND METHODS Study Design This study included 5,516 normally fertilized zygotes, which were obtained from 605 patients undergoing intracytoplasmic sperm injection (ICSI) cycles, between January 2011 and August 2012, in which the ET was performed on day 5 of embryo development. The oocytes were evaluated immediately before the sperm injection, and the embryos were evaluated 16–18 hours after ICSI and on days 2, 3, and 5 of development. All of the cases with severe spermatogenic alteration, including frozen and surgically retrieved sperm, were excluded from the study. The blastocysts were graded according to the Gardner and Schoolcraft system (33). The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm (TE) cells, were correlated with oocyte dysmorphisms. In cycles in which the implantation rate was either 100% (n ¼ 190) or 0 (n ¼ 339), the effect of oocyte defects on the pregnancy and miscarriage rate was analysed. Written informed consent, in which patients agreed to share the outcomes of their cycles for research purposes, was obtained, and the local institutional review board approved the study.
Controlled Ovarian Stimulation Controlled ovarian stimulation was achieved by using recombinant FSH (Gonal-F; Serono) as a daily dose, starting on day 3 of the cycle. Pituitary blockage was performed by using a GnRH antagonist (Cetrotide; Serono), starting when at least one follicle R14 mm was visualized. Follicular growth was monitored using transvaginal ultrasound examination starting on day 4 of gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG (Ovidrel; Serono) was administered to trigger the final follicular maturation. The oocytes were collected 35 hours after hCG administration through transvaginal ultrasound ovum pick-up.
Preparation of Oocytes Retrieved oocytes were maintained in culture medium (Global for fertilization; LifeGlobal) supplemented with 10% protein VOL. 100 NO. 3 / SEPTEMBER 2013
supplement (LGPS; LifeGlobal) and covered with paraffin oil (Paraffin oil P.G.; LifeGlobal) for 2 to 3 hours before the removal of cumulus cells. The surrounding cumulus cells were removed after exposure to an N-2hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES)buffered medium containing hyaluronidase (80 IU/mL; LifeGlobal). The remaining cumulus cells were mechanically removed by gently pipetting with a hand-drawn Pasteur pipette (Humagen Fertility Diagnostics). Oocyte morphology was assessed just before sperm injection (4 hours after retrieval) using an inverted Nikon Diaphot microscope (Eclipse TE 300) with a Hoffmann modulation contrast system under 400 magnification. The following oocyte dysmorphisms were recorded: [1] cytoplasmic granularity, [2] cytoplasmic color, [3] vacuoles in the ooplasm, [4] aggregates of smooth endoplasmic reticulum clusters (ERC) in the ooplasm, [5] large PVS, [6] PVS granularity, [7] fragmented polar body (PB), [8] ZP abnormalities, and [9] oocyte shape abnormalities. Oocytes that had released the first PB were considered mature and were used for ICSI.
Intracytoplasmic Sperm Injection Intracytoplasmic sperm injection was performed in a microinjection dish prepared with 4-mL droplets of buffered medium (Global w/HEPES; LifeGlobal) and covered with paraffin oil on the heated stage of an inverted microscope (37.0 C 0.5 C). Approximately 16 hours after ICSI, fertilization was confirmed by the presence of two pronuclei and the extrusion of the second PB. Embryos were maintained in a 50-mL drop of culture medium (Global) supplemented with 10% protein supplement and covered with paraffin oil in a humidified atmosphere under 6% CO2 at 37 C for 3 days.
Embryo Morphology Evaluation Embryo morphology was assessed at zygote stage (16–18 hours after ICSI) and on the mornings of days 2, 3, and 5 of embryo development using an inverted Nikon Diaphot microscope (Eclipse TE 300) with a Hoffmann modulation contrast system under 400 magnification. Immediately before ET the embryo morphology was also assessed. To evaluate the cleavage-stage morphology, the following parameters were recorded: the number of blastomeres, the percentage of fragmentation, the variation in blastomere symmetry, the presence of multinucleation, and defects in the ZP and cytoplasm. The high-quality cleavagestage embryos were defined as those with all of the following characteristics: 4 cells on day 2 or 8–10 cells on day 3, <15% fragmentation, symmetric blastomeres, the absence of multinucleation, colorless cytoplasm with moderate granulation and no inclusions, the absence of PVS granularity, and the absence of ZP dysmorphism. Embryos lacking any of these characteristics were considered to be of low quality. To evaluate the blastocyst morphology, embryos were given a numerical score from 1 to 6 according to their degree of expansion and hatching status, as follows: 1, an early blastocyst with a blastocoel that is less than half the volume 749
ORIGINAL ARTICLE: ASSISTED REPRODUCTION of the embryo; 2, a blastocyst with a blastocoel that is greater than half the volume of the embryo; 3, a full blastocyst with a blastocoel that completely fills the embryo; 4, an expanded blastocyst; 5, a hatching blastocyst; and 6, a hatched blastocyst. The ICM of full, expanded, hatching, and hatched blastocysts was classified as either high quality (tightly packed with many cells) or low quality (loosely grouped with several or few cells). Similarly, the TE cells were also classified as either high quality (many cells forming a cohesive epithelium) or low quality (few cells forming a loose epithelium or very few cells).
TABLE 1 Incidence of extracytoplasmic and cytoplasmic oocyte abnormalities in ICSI cycles.
Response variable Cytoplasmic
Extracytoplasmic
Statistical Analyses Ordinal regression analyses were performed to study the influence of oocyte dysmorphisms on the blastocysts' degree of expansion and hatching status. Binary regression analysis was performed to evaluate the influence of oocyte dysmorphisms on ICM and TE cell quality and on the pregnancy chance. All regression analysis was adjusted for sperm concentration, motility and morphology, maternal age, number of retrieved oocytes, total dose of FSH used for ovarian stimulation, and fertilization rate, because these variables were considered potential confounders in the association between the factors evaluated and ICSI outcomes. Results were expressed as odds ratios (ORs), regression coefficients, 95% confidence intervals (CIs), and P values. Results were considered to be significant at the 5% critical level (P< .05). Data analysis was carried out using Minitab Statistical Software (version 14, Minitab Inc.).
RESULTS The patients' characteristics are described as the mean SD and were as follows: maternal age: 33.5 10.3 years; paternal age: 37.6 18 years; total dose of FSH administered for ovarian stimulation: 2,045 715 IU; serum 17b-estradiol levels on the ovulation trigger day: 2,545.9 1,445 pg/mL; number of aspirated follicles: 12.8 12.7; and number of retrieved oocytes: 9.5 11.3. A total of 5,516 oocytes/embryos were evaluated. Of these oocytes, 3,345 (60.4%) had at least one morphologic abnormality. Cytoplasmic and extracytoplasmic
Predictor variable
Incidence per evaluated embryo, n (%)
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
2,173 (39.4) 358 (6.5) 325 (5.9) 375 (6.8) 1,097 (19.9) 568 (10.3) 507 (9.2) 292 (5.3)
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
abnormalities were seen in 1,390 (41.5%) and 1,955 (58.4%) of the oocytes, respectively. The incidence of oocyte dysmorphisms per cycle was 97.8%. The incidence of abnormal oocyte morphotypes is described in Table 1. A total of 2,920 (52.9%) reached the blastocyst stage. Of the embryos that reached the blastocyst stage, 858 (29.4%) were grade 1; 584 (20.0%) were grade 2; 433 (14.8%) were grade 3; 262 (9.0%) were grade 4; 496 (17.0%) were grade 5; and 287 (9.8%) were grade 6. The ordinal regression model showed that the blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth ERC, large PVS, and shape abnormalities (Table 2). The binary regression model demonstrated that the presence of a large PVS decreased by 15% the ICM quality and by 50% the TE cell quality. Aggregates of smooth ERC accounted for a 35% decrease in the ICM quality (Table 3), a 67% decrease in the pregnancy chance, and a 20% increase in the miscarriage risk (Table 4).
DISCUSSION The transfer of human embryos at the blastocyst stage is becoming increasingly popular as a tool for infertility therapy (34). Even though many advantages of extended embryo culture until the blastocyst stage have been reported, extended embryo culture may lead to an ET cancellation due to an insufficient number of embryos available for transfer.
TABLE 2 Ordinal regression analysis of oocyte dysmorphisms that may affect the blastocysts' degree of expansion and hatching status. Response variable Degree of expansion and hatching status
a
Predictor variable
P value
OR
CI: lower
CI: upper
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
.084 .083 .178 .020a < .001a .955 .235 .002a
1.33 1.88 1.21 0.61 0.80 1.45 0.84 0.75
0.96 0.92 0.92 0.40 0.71 1.22 0.64 0.24
1.84 3.82 1.60 0.93 0.91 0.91 1.12 0.95
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
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TABLE 3 Binary regression analysis of oocyte dysmorphisms that may affect the blastocysts' inner cell mass and trophectoderm quality. Response variable ICM quality
TE cell quality
a
Predictor variable
P value
OR
CI: lower
CI: upper
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities. Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities.
.998 .708 .983 .005a .045a .158 .122 .763 .075 .153 .746 .731 < .001a .210 .333 .618
1.15 1.87 1.01 0.65 0.85 0.53 2.09 0.74 0.50 0.24 0.92 1.13 0.50 1.15 1.27 0.84
0.95 0.92 0.31 0.12 0.63 0.22 0.82 0.10 0.24 0.03 0.57 0.55 0.36 0.93 0.79 0.41
2.15 2.68 3.28 0.85 0.98 1.28 2.35 3.01 0.98 1.71 1.49 2.33 0.71 1.42 1.42 1.70
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
There is a general tendency to disregard oocyte morphology when selecting an embryo or blastocyst for transfer. However, oocyte quality is a key limiting factor in female fertility, reflecting the intrinsic developmental potential of an oocyte, and plays a crucial role not only in fertilization but also in subsequent development (35). According to some authors, the phenotype of the adult-stage offspring is considerably defined by the quality of the oocytes from which they are derived (36). In the present study we evaluated the predictive value of oocyte morphology on blastocyst formation and morphology. Our results showed a negative impact of specific intracytoplasmic and extracytoplasmic oocyte defects on the three blastocyst quality parameters that were evaluated: the blastocysts' degree of expansion and hatching status, and ICM and TE cell quality. It has been reported that the development of intracytoplasmic and extracytoplasmic anomalies during the
maturation process may lead to fertilization failure (10, 15, 37), chromosome aneuploidy (13, 38), and developmental impairment of the embryo despite normal fertilization (2, 14, 39–41). Analysis of the literature, however, shows that the effect of oocyte morphologic deviations after ICSI remains controversial (6, 42, 43). Our evidence suggests a possible negative influence of the presence of large PVS on the blastocysts' degree of expansion and hatching status, ICM quality, and TE cell quality. Conversely, Balaban and Urman (43) suggested that extracytoplasmic defects should be considered only as phenotypic deviations resulting from the heterogeneity of the oocytes retrieved. In fact, no correlations between increased PVS and further developmental characteristics were previously reported (6, 10, 42). However, Ten et al. (44) found an association between increased PVS and decreased embryo quality. Large PVS
TABLE 4 Binary regression analysis of oocyte dysmorphisms that may affect the pregnancy chance and miscarriage risk. Response variable
Predictor variable
P value
OR
CI: lower
CI: upper
Pregnancy chance
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities. Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
.356 .498 .354 .048a .985 .645 .485 .365 .356 .658 .564 .035a .458 .359 .976 .694
0.57 0.42 1.01 0.33 0.98 1.15 1.36 1.02. 0.85 0.99 0.56 1.23 1.03 1.25 1.12 1.15.
0.24 0.14 0.33 0.99 0.63 0.93 1.13. 0.93 0.65 0.85 0.12 1.05 0.36 0.89 1.03. 0.75
1.03 1.52 1.74 0.98 1.61 1.42 1.82 1.89 1.33 1.32 1.01 1.45 1.56 1.65 1.95 1.35
Miscarriage risk
a
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION was also correlated with low fertilization rates and compromised pronuclear morphology but had no further effect on embryo quality (15). Additionally, in a recent meta-analysis from our group (11) it was demonstrated that the probability of an oocyte becoming fertilized is significantly reduced by the presence of a large PVS. According to Miao et al. (45), a series of morphologic and cellular changes occur during the process of oocyte aging, including PVS increasing, and it is suggested that fertilization of aged oocytes not only affects pre- and postimplantation embryo development but also the later life of the offspring (45). Our findings also demonstrated a negative influence of the presence of aggregates of smooth ERC on the blastocysts' degree of expansion and hatching status and ICM quality. It is presumed that these clusters arise by dilatation and fusion of smooth endoplasmic reticulum saccules during the gamete's maturational process (38). It has also been suggested that ERC arise as a consequence of ovary hyperstimulation, because these forms have never been observed in germinal vesicle oocytes aspirated from antral follicles in nonstimulated ovaries (5). Although it has been suggested that these dysmorphic oocytes present a high aneuploidy rate (18%–37%) (38), the organization of the meiotic spindle is not affected by the large ERC (7). The relatively low frequency of aggregates of smooth ERC has prevented several authors from analyzing this oocyte dysmorphism separately, and the majority of studies did not evaluate vacuolization, refractile bodies, or aggregates of smooth ERC as individual features (2, 4, 10, 16, 18, 46). These features have been characterised as cytoplasmic inclusions. According to some investigators, cytoplasmic inclusions do not seem to affect fertilization, embryo quality, or implantation rates (6, 10). Others, however, have reported decreased fertilization and embryo development (2, 47). In a previous report in which the presence of smooth ERC was individually registered and its predictive value was individually analyzed, an association between the presence of smooth ERC and lower chances of successful fertilization and pregnancy was demonstrated (48). Otsuki et al. (14) reported that the presence of ERC is associated with lower chances of successful pregnancy, even in ERC-negative oocytes from the same cohort that are transferred along with the ERC-positive oocytes. Besides the lower chance of success after IVF, the presence of ERC may also be associated with fetal anomalies (49). According to Ebner et al. (41), once the oocyte was fertilized, no further limitation in development was recognizable up to day 3 and day 4. On day 5, however, significantly fewer embryos reached the blastocyst stage if they were derived from ERC-positive oocytes. The effects of oocyte morphologic abnormalities on pregnancy rates were investigated in a few studies (7, 8, 17). These comparisons are applicable in the context of single embryo transfer; however, this was not the case of any of these studies. To evaluate the effect of oocyte defects on the pregnancy fate, only cycles in which the implantation rate was either 100% or 0 were evaluated separately. The results 752
demonstrated that besides the blastocyst quality, the presence of ERC is determinant of increased odds of pregnancy. In our study the influence of other cytoplasmic defects on blastocyst quality, such as cytoplasmic granularity, was not noted. Conversely, Rienzi et al. (15) described a negative influence of diffused cytoplasmic granularity on pronuclear morphology and day-2 embryo quality. However, in a systematic review of the literature regarding the predictive value of oocyte morphology in IVF, the same author analyzed 50 articles published in the past 15 years and concluded that their analysis has produced contradicting results and did not entirely support the average opinion about the features of ‘‘good’’ and ‘‘bad’’ quality and respective developmental competence. Moreover, the study underlined the importance of more intensive research to reach a consensus and exploit fully the predictive potential of morphologic examination (50). One particular dysmorphism that definitely affects oocyte structure is a shape anomaly. However, a case report indicates that oocytes with an ovoid shape may result in successful pregnancy (51). In the present study, shape anomaly was correlated with decreased blastocyst development. This finding is in line with previous reports demonstrating that ovoid-shaped oocytes are associated with delays in in vitro parameters (52). After fertilization, ZP assists the oviductal transport of the embryo and plays a role in protecting the integrity of the developing embryo (53). A spherical shape of the ZP ensures maximal contact between the blastomeres; in this scenario, the embryo will cleave as expected, giving a crosswise arrangement of four cells with intense contact that will facilitate compaction owing to the larger number of tight junctions available (52). Thus, it is expected that cleaving embryos derived from ovoid oocytes face delayed compaction and blastocyst formation, as observed in the present study. In summary, the results presented here suggest that the individual identification of oocyte dysmorphisms may be a prognostic tool for blastocyst development and quality. Patients in whom specific oocyte defects are detected may have the cycle outcome compromised and not benefit from postponing ET until day 5 of development. In these cases, cleavage-stage ET would be a better approach.
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Objective: To identify the association between oocyte dysmorphisms and blastocyst developmental competence. Design: Case–control study. Setting: Private assisted reproduction center. Patient(s): This study included 5,516 embryos, which were obtained from 605 patients undergoing intracytoplasmic sperm injection cycles in which ET was performed on day 5 of embryo development. Intervention(s): Intracytoplasmic sperm injection and ET. Main Outcome Measure(s): The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm cells, were correlated with oocyte dysmorphisms. Result(s): Of the 5,516 embryos evaluated, 2,920 (52.9%) reached the blastocyst stage. The blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth endoplasmic reticulum clusters, large perivitelline space, and shape abnormalities. The presence of a large perivitelline space also decreased the ICM and trophectoderm cell quality. Aggregates of smooth endoplasmic reticulum clusters also decreased the ICM quality, pregnancy rate, and the miscarriage rate. Conclusion(s): The results presented here suggest that the individual identification of oocyte dymorphisms may be a prognostic tool for blastocyst development and quality. However, the results do not diminish the importance of cleavage-stage embryo morphology assessment to predict blastocyst development competence. (Fertil SterilÒ 2013;100:748–54. Use your smartphone Ó2013 by American Society for Reproductive Medicine.) to scan this QR code Key Words: Oocyte, blastocyst, perivitelline space, smooth endoplasmic reticulum, oocyte and connect to the shape Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/bragadpdaf-oocyte-dysmorphisms-blastocyst-formation/
O
ocyte quality has been regarded as a variable that influences the implantation potential of derived embryos. To date there have been many published reports on the impact of oocyte morphology on embryo development (1–4). The most frequently observed oocyte morphologic variations are cytoplasmic, including changes in color, granularity, and homogeneity, as well as cytoplasmic incorporations.
Extracytoplasmic variations are deviations from normal perivitelline space (PVS), zona pellucida (ZP) color, and oocyte shape (5). However, the predictive value of the criteria used in these previous studies is still controversial (2, 6–19). An extended embryo culture and the transfer of blastocyst-stage embryos are associated with increased implantation rates compared with the transfer of cleavage-stage embryos
Received March 1, 2013; revised May 9, 2013; accepted May 15, 2013; published online June 12, 2013. D.P.A.F.B. has nothing to disclose. A.S.S. has nothing to disclose. R.d.C.S.F. has nothing to disclose. R.B.M. has nothing to disclose. A.I. has nothing to disclose. E.B. has nothing to disclose. ~o Assistida, Av. BrigadeReprint requests: Edson Borges Jr., M.D., Ph.D., Fertility, Centro de Fertilizac¸a ^ nio 4545, 01401-002 Sa ~o Paulo, Sa ~o Paulo, Brazil (E-mail: [email protected]). iro Luis Anto Fertility and Sterility® Vol. 100, No. 3, September 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.05.021 748
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(20, 21). A prolonged culture period allows for a better selection of more-advanced embryos that are not arrested because the laboratory assessment is undertaken after the embryonic genome has begun to be expressed (22). In addition, it has been reported that better synchronization between endometrium function and embryo development is possible with blastocyst-stage ETs (23, 24). Another potential benefit of blastocyst culture would be that at day 5, uterine contractility is decreased as compared with days 2 or 3 (25). However, the proportion of embryos that reach the blastocyst stage is still unpredictable (26–28), and some assisted reproduction centers are reluctant to adopt extended embryo VOL. 100 NO. 3 / SEPTEMBER 2013
Fertility and Sterility® cultures to avoid any ET cancellation due to an unpredictable rate of blastocyst development. Various factors may be responsible for this variation in the rate of blastocyst formation. Considering the vital role played by the oocyte in the developmental process, the noninvasive identification of oocyte dysmorphisms before fertilization would be extremely useful in predicting the rate of blastocyst formation. There is growing evidence in animal studies to support the idea that recurrent failure in assisted reproduction could be explained by low oocyte quality (29–32). However, although animal studies seem to support an association between oocyte and blastocyst quality, human studies are scarce. Therefore, the goal of the present study was to identify the association between oocyte dysmorphisms and blastocyst developmental competence.
MATERIALS AND METHODS Study Design This study included 5,516 normally fertilized zygotes, which were obtained from 605 patients undergoing intracytoplasmic sperm injection (ICSI) cycles, between January 2011 and August 2012, in which the ET was performed on day 5 of embryo development. The oocytes were evaluated immediately before the sperm injection, and the embryos were evaluated 16–18 hours after ICSI and on days 2, 3, and 5 of development. All of the cases with severe spermatogenic alteration, including frozen and surgically retrieved sperm, were excluded from the study. The blastocysts were graded according to the Gardner and Schoolcraft system (33). The morphologic characteristics of the blastocysts, namely the degree of blastocoel expansion and hatching status, the size and compactness of the inner cell mass (ICM), and the cohesiveness and number of trophectoderm (TE) cells, were correlated with oocyte dysmorphisms. In cycles in which the implantation rate was either 100% (n ¼ 190) or 0 (n ¼ 339), the effect of oocyte defects on the pregnancy and miscarriage rate was analysed. Written informed consent, in which patients agreed to share the outcomes of their cycles for research purposes, was obtained, and the local institutional review board approved the study.
Controlled Ovarian Stimulation Controlled ovarian stimulation was achieved by using recombinant FSH (Gonal-F; Serono) as a daily dose, starting on day 3 of the cycle. Pituitary blockage was performed by using a GnRH antagonist (Cetrotide; Serono), starting when at least one follicle R14 mm was visualized. Follicular growth was monitored using transvaginal ultrasound examination starting on day 4 of gonadotropin administration. When adequate follicular growth and serum E2 levels were observed, recombinant hCG (Ovidrel; Serono) was administered to trigger the final follicular maturation. The oocytes were collected 35 hours after hCG administration through transvaginal ultrasound ovum pick-up.
Preparation of Oocytes Retrieved oocytes were maintained in culture medium (Global for fertilization; LifeGlobal) supplemented with 10% protein VOL. 100 NO. 3 / SEPTEMBER 2013
supplement (LGPS; LifeGlobal) and covered with paraffin oil (Paraffin oil P.G.; LifeGlobal) for 2 to 3 hours before the removal of cumulus cells. The surrounding cumulus cells were removed after exposure to an N-2hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES)buffered medium containing hyaluronidase (80 IU/mL; LifeGlobal). The remaining cumulus cells were mechanically removed by gently pipetting with a hand-drawn Pasteur pipette (Humagen Fertility Diagnostics). Oocyte morphology was assessed just before sperm injection (4 hours after retrieval) using an inverted Nikon Diaphot microscope (Eclipse TE 300) with a Hoffmann modulation contrast system under 400 magnification. The following oocyte dysmorphisms were recorded: [1] cytoplasmic granularity, [2] cytoplasmic color, [3] vacuoles in the ooplasm, [4] aggregates of smooth endoplasmic reticulum clusters (ERC) in the ooplasm, [5] large PVS, [6] PVS granularity, [7] fragmented polar body (PB), [8] ZP abnormalities, and [9] oocyte shape abnormalities. Oocytes that had released the first PB were considered mature and were used for ICSI.
Intracytoplasmic Sperm Injection Intracytoplasmic sperm injection was performed in a microinjection dish prepared with 4-mL droplets of buffered medium (Global w/HEPES; LifeGlobal) and covered with paraffin oil on the heated stage of an inverted microscope (37.0 C 0.5 C). Approximately 16 hours after ICSI, fertilization was confirmed by the presence of two pronuclei and the extrusion of the second PB. Embryos were maintained in a 50-mL drop of culture medium (Global) supplemented with 10% protein supplement and covered with paraffin oil in a humidified atmosphere under 6% CO2 at 37 C for 3 days.
Embryo Morphology Evaluation Embryo morphology was assessed at zygote stage (16–18 hours after ICSI) and on the mornings of days 2, 3, and 5 of embryo development using an inverted Nikon Diaphot microscope (Eclipse TE 300) with a Hoffmann modulation contrast system under 400 magnification. Immediately before ET the embryo morphology was also assessed. To evaluate the cleavage-stage morphology, the following parameters were recorded: the number of blastomeres, the percentage of fragmentation, the variation in blastomere symmetry, the presence of multinucleation, and defects in the ZP and cytoplasm. The high-quality cleavagestage embryos were defined as those with all of the following characteristics: 4 cells on day 2 or 8–10 cells on day 3, <15% fragmentation, symmetric blastomeres, the absence of multinucleation, colorless cytoplasm with moderate granulation and no inclusions, the absence of PVS granularity, and the absence of ZP dysmorphism. Embryos lacking any of these characteristics were considered to be of low quality. To evaluate the blastocyst morphology, embryos were given a numerical score from 1 to 6 according to their degree of expansion and hatching status, as follows: 1, an early blastocyst with a blastocoel that is less than half the volume 749
ORIGINAL ARTICLE: ASSISTED REPRODUCTION of the embryo; 2, a blastocyst with a blastocoel that is greater than half the volume of the embryo; 3, a full blastocyst with a blastocoel that completely fills the embryo; 4, an expanded blastocyst; 5, a hatching blastocyst; and 6, a hatched blastocyst. The ICM of full, expanded, hatching, and hatched blastocysts was classified as either high quality (tightly packed with many cells) or low quality (loosely grouped with several or few cells). Similarly, the TE cells were also classified as either high quality (many cells forming a cohesive epithelium) or low quality (few cells forming a loose epithelium or very few cells).
TABLE 1 Incidence of extracytoplasmic and cytoplasmic oocyte abnormalities in ICSI cycles.
Response variable Cytoplasmic
Extracytoplasmic
Statistical Analyses Ordinal regression analyses were performed to study the influence of oocyte dysmorphisms on the blastocysts' degree of expansion and hatching status. Binary regression analysis was performed to evaluate the influence of oocyte dysmorphisms on ICM and TE cell quality and on the pregnancy chance. All regression analysis was adjusted for sperm concentration, motility and morphology, maternal age, number of retrieved oocytes, total dose of FSH used for ovarian stimulation, and fertilization rate, because these variables were considered potential confounders in the association between the factors evaluated and ICSI outcomes. Results were expressed as odds ratios (ORs), regression coefficients, 95% confidence intervals (CIs), and P values. Results were considered to be significant at the 5% critical level (P< .05). Data analysis was carried out using Minitab Statistical Software (version 14, Minitab Inc.).
RESULTS The patients' characteristics are described as the mean SD and were as follows: maternal age: 33.5 10.3 years; paternal age: 37.6 18 years; total dose of FSH administered for ovarian stimulation: 2,045 715 IU; serum 17b-estradiol levels on the ovulation trigger day: 2,545.9 1,445 pg/mL; number of aspirated follicles: 12.8 12.7; and number of retrieved oocytes: 9.5 11.3. A total of 5,516 oocytes/embryos were evaluated. Of these oocytes, 3,345 (60.4%) had at least one morphologic abnormality. Cytoplasmic and extracytoplasmic
Predictor variable
Incidence per evaluated embryo, n (%)
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
2,173 (39.4) 358 (6.5) 325 (5.9) 375 (6.8) 1,097 (19.9) 568 (10.3) 507 (9.2) 292 (5.3)
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
abnormalities were seen in 1,390 (41.5%) and 1,955 (58.4%) of the oocytes, respectively. The incidence of oocyte dysmorphisms per cycle was 97.8%. The incidence of abnormal oocyte morphotypes is described in Table 1. A total of 2,920 (52.9%) reached the blastocyst stage. Of the embryos that reached the blastocyst stage, 858 (29.4%) were grade 1; 584 (20.0%) were grade 2; 433 (14.8%) were grade 3; 262 (9.0%) were grade 4; 496 (17.0%) were grade 5; and 287 (9.8%) were grade 6. The ordinal regression model showed that the blastocysts' degree of expansion and hatching status were decreased by the presence of aggregates of smooth ERC, large PVS, and shape abnormalities (Table 2). The binary regression model demonstrated that the presence of a large PVS decreased by 15% the ICM quality and by 50% the TE cell quality. Aggregates of smooth ERC accounted for a 35% decrease in the ICM quality (Table 3), a 67% decrease in the pregnancy chance, and a 20% increase in the miscarriage risk (Table 4).
DISCUSSION The transfer of human embryos at the blastocyst stage is becoming increasingly popular as a tool for infertility therapy (34). Even though many advantages of extended embryo culture until the blastocyst stage have been reported, extended embryo culture may lead to an ET cancellation due to an insufficient number of embryos available for transfer.
TABLE 2 Ordinal regression analysis of oocyte dysmorphisms that may affect the blastocysts' degree of expansion and hatching status. Response variable Degree of expansion and hatching status
a
Predictor variable
P value
OR
CI: lower
CI: upper
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
.084 .083 .178 .020a < .001a .955 .235 .002a
1.33 1.88 1.21 0.61 0.80 1.45 0.84 0.75
0.96 0.92 0.92 0.40 0.71 1.22 0.64 0.24
1.84 3.82 1.60 0.93 0.91 0.91 1.12 0.95
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
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TABLE 3 Binary regression analysis of oocyte dysmorphisms that may affect the blastocysts' inner cell mass and trophectoderm quality. Response variable ICM quality
TE cell quality
a
Predictor variable
P value
OR
CI: lower
CI: upper
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities. Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities.
.998 .708 .983 .005a .045a .158 .122 .763 .075 .153 .746 .731 < .001a .210 .333 .618
1.15 1.87 1.01 0.65 0.85 0.53 2.09 0.74 0.50 0.24 0.92 1.13 0.50 1.15 1.27 0.84
0.95 0.92 0.31 0.12 0.63 0.22 0.82 0.10 0.24 0.03 0.57 0.55 0.36 0.93 0.79 0.41
2.15 2.68 3.28 0.85 0.98 1.28 2.35 3.01 0.98 1.71 1.49 2.33 0.71 1.42 1.42 1.70
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
There is a general tendency to disregard oocyte morphology when selecting an embryo or blastocyst for transfer. However, oocyte quality is a key limiting factor in female fertility, reflecting the intrinsic developmental potential of an oocyte, and plays a crucial role not only in fertilization but also in subsequent development (35). According to some authors, the phenotype of the adult-stage offspring is considerably defined by the quality of the oocytes from which they are derived (36). In the present study we evaluated the predictive value of oocyte morphology on blastocyst formation and morphology. Our results showed a negative impact of specific intracytoplasmic and extracytoplasmic oocyte defects on the three blastocyst quality parameters that were evaluated: the blastocysts' degree of expansion and hatching status, and ICM and TE cell quality. It has been reported that the development of intracytoplasmic and extracytoplasmic anomalies during the
maturation process may lead to fertilization failure (10, 15, 37), chromosome aneuploidy (13, 38), and developmental impairment of the embryo despite normal fertilization (2, 14, 39–41). Analysis of the literature, however, shows that the effect of oocyte morphologic deviations after ICSI remains controversial (6, 42, 43). Our evidence suggests a possible negative influence of the presence of large PVS on the blastocysts' degree of expansion and hatching status, ICM quality, and TE cell quality. Conversely, Balaban and Urman (43) suggested that extracytoplasmic defects should be considered only as phenotypic deviations resulting from the heterogeneity of the oocytes retrieved. In fact, no correlations between increased PVS and further developmental characteristics were previously reported (6, 10, 42). However, Ten et al. (44) found an association between increased PVS and decreased embryo quality. Large PVS
TABLE 4 Binary regression analysis of oocyte dysmorphisms that may affect the pregnancy chance and miscarriage risk. Response variable
Predictor variable
P value
OR
CI: lower
CI: upper
Pregnancy chance
Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities. Cytoplasmic granularity Cytoplasmic color Vacuoles in the ooplasm Aggregates of smooth ERC Large PVS PVS granularity ZP abnormalities Shape abnormalities
.356 .498 .354 .048a .985 .645 .485 .365 .356 .658 .564 .035a .458 .359 .976 .694
0.57 0.42 1.01 0.33 0.98 1.15 1.36 1.02. 0.85 0.99 0.56 1.23 1.03 1.25 1.12 1.15.
0.24 0.14 0.33 0.99 0.63 0.93 1.13. 0.93 0.65 0.85 0.12 1.05 0.36 0.89 1.03. 0.75
1.03 1.52 1.74 0.98 1.61 1.42 1.82 1.89 1.33 1.32 1.01 1.45 1.56 1.65 1.95 1.35
Miscarriage risk
a
Statistically significant.
Braga. Oocyte defects and blastocyst quality. Fertil Steril 2013.
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ORIGINAL ARTICLE: ASSISTED REPRODUCTION was also correlated with low fertilization rates and compromised pronuclear morphology but had no further effect on embryo quality (15). Additionally, in a recent meta-analysis from our group (11) it was demonstrated that the probability of an oocyte becoming fertilized is significantly reduced by the presence of a large PVS. According to Miao et al. (45), a series of morphologic and cellular changes occur during the process of oocyte aging, including PVS increasing, and it is suggested that fertilization of aged oocytes not only affects pre- and postimplantation embryo development but also the later life of the offspring (45). Our findings also demonstrated a negative influence of the presence of aggregates of smooth ERC on the blastocysts' degree of expansion and hatching status and ICM quality. It is presumed that these clusters arise by dilatation and fusion of smooth endoplasmic reticulum saccules during the gamete's maturational process (38). It has also been suggested that ERC arise as a consequence of ovary hyperstimulation, because these forms have never been observed in germinal vesicle oocytes aspirated from antral follicles in nonstimulated ovaries (5). Although it has been suggested that these dysmorphic oocytes present a high aneuploidy rate (18%–37%) (38), the organization of the meiotic spindle is not affected by the large ERC (7). The relatively low frequency of aggregates of smooth ERC has prevented several authors from analyzing this oocyte dysmorphism separately, and the majority of studies did not evaluate vacuolization, refractile bodies, or aggregates of smooth ERC as individual features (2, 4, 10, 16, 18, 46). These features have been characterised as cytoplasmic inclusions. According to some investigators, cytoplasmic inclusions do not seem to affect fertilization, embryo quality, or implantation rates (6, 10). Others, however, have reported decreased fertilization and embryo development (2, 47). In a previous report in which the presence of smooth ERC was individually registered and its predictive value was individually analyzed, an association between the presence of smooth ERC and lower chances of successful fertilization and pregnancy was demonstrated (48). Otsuki et al. (14) reported that the presence of ERC is associated with lower chances of successful pregnancy, even in ERC-negative oocytes from the same cohort that are transferred along with the ERC-positive oocytes. Besides the lower chance of success after IVF, the presence of ERC may also be associated with fetal anomalies (49). According to Ebner et al. (41), once the oocyte was fertilized, no further limitation in development was recognizable up to day 3 and day 4. On day 5, however, significantly fewer embryos reached the blastocyst stage if they were derived from ERC-positive oocytes. The effects of oocyte morphologic abnormalities on pregnancy rates were investigated in a few studies (7, 8, 17). These comparisons are applicable in the context of single embryo transfer; however, this was not the case of any of these studies. To evaluate the effect of oocyte defects on the pregnancy fate, only cycles in which the implantation rate was either 100% or 0 were evaluated separately. The results 752
demonstrated that besides the blastocyst quality, the presence of ERC is determinant of increased odds of pregnancy. In our study the influence of other cytoplasmic defects on blastocyst quality, such as cytoplasmic granularity, was not noted. Conversely, Rienzi et al. (15) described a negative influence of diffused cytoplasmic granularity on pronuclear morphology and day-2 embryo quality. However, in a systematic review of the literature regarding the predictive value of oocyte morphology in IVF, the same author analyzed 50 articles published in the past 15 years and concluded that their analysis has produced contradicting results and did not entirely support the average opinion about the features of ‘‘good’’ and ‘‘bad’’ quality and respective developmental competence. Moreover, the study underlined the importance of more intensive research to reach a consensus and exploit fully the predictive potential of morphologic examination (50). One particular dysmorphism that definitely affects oocyte structure is a shape anomaly. However, a case report indicates that oocytes with an ovoid shape may result in successful pregnancy (51). In the present study, shape anomaly was correlated with decreased blastocyst development. This finding is in line with previous reports demonstrating that ovoid-shaped oocytes are associated with delays in in vitro parameters (52). After fertilization, ZP assists the oviductal transport of the embryo and plays a role in protecting the integrity of the developing embryo (53). A spherical shape of the ZP ensures maximal contact between the blastomeres; in this scenario, the embryo will cleave as expected, giving a crosswise arrangement of four cells with intense contact that will facilitate compaction owing to the larger number of tight junctions available (52). Thus, it is expected that cleaving embryos derived from ovoid oocytes face delayed compaction and blastocyst formation, as observed in the present study. In summary, the results presented here suggest that the individual identification of oocyte dysmorphisms may be a prognostic tool for blastocyst development and quality. Patients in whom specific oocyte defects are detected may have the cycle outcome compromised and not benefit from postponing ET until day 5 of development. In these cases, cleavage-stage ET would be a better approach.
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