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Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments
G Model REPBIO 236 No. of Pages 8
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Original article
Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments Somayyeh Safaria , Mohammad Ali Khalilia,* , Zeinab Barekatib , Iman Halvaeic , Morteza Anvaria , Stefania A. Nottolad a
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Genome Plasticity, Department of Biomedicine, University of Basel, Switzerland c Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran d Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Rome, Italy b
A R T I C L E I N F O
Article history: Received 19 February 2017 Received in revised form 6 April 2017 Accepted 6 May 2017 Available online xxx Keywords: Cosmetic micromanipulation Fragment removal Vitrified-warmed embryos Pregnancy outcome Transmission electron microscopy
A B S T R A C T
The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n = 32), sham (n = 32) and control (n = 32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P = 0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrifiedwarmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments. © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
1. Introduction Fragmentation or generation of small anuclear cytoplasmic fragments is a common feature of human developing embryos [1,2]. In various embryo grading systems, the degree of fragmentation and the developmental stage of embryos are defined as the most common used criteria for selecting a high-quality embryo for
* Corresponding author at: Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Bouali Ave, Safaeiyeh, Yazd, 89168-77391, Iran. E-mail addresses: [email protected] (S. Safari), [email protected] (M.A. Khalili), [email protected] (Z. Barekati), [email protected] (I. Halvaei), [email protected] (M. Anvari), [email protected] (S.A. Nottola).
transfer following in vitro fertilization (IVF) [3]. In spite of the unknown mechanism underlying the generation of fragments, it is clear that this phenomenon is associated with a deleterious assisted reproductive technology (ART) outcomes [4–8]. For example, further development of fragmented embryos is impaired, often followed by cleavage arrest and degeneration. In addition, transfer of these embryos showed limited developmental potential and rarely resulted in successful pregnancy [5,9]. Therefore, fragmentation is an important biomarker for poor pregnancy outcomes [1,10–12]. It has been proposed that cytoplasmic fragmentation induces apoptosis and limits blastomeres cleavage rate. Therefore, removal of these fragments may improve cell division and implantation [13]. In addition to fragments formation in embryos, the perivitelline space (PVS) in fragmented embryos may be filled with other debris,
http://dx.doi.org/10.1016/j.repbio.2017.05.003 1642-431X/© 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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such as coarse granules. Farhi and co-workers showed that this dysmorphism had a negative impact on ART outcome, and transfer of embryos generated from oocytes with PVS coarse granulation resulted in significant lower implantation and pregnancy rates [14]. Recently Halvaei et al. showed that removal of fragments and coarse granules (cosmetic micromanipulation) from low-grade embryos improved pregnancy rates in patients with implantation failure in fresh cycles [15]. Frozen embryo transfer (FET) cycles constitute approximately 20% of all performed ART cycles in assisted reproduction clinics [16]. It is important to note that using frozen embryos in ART cycles is less invasive for the mother rather than starting a new cycle of hormone therapy to stimulate ovaries and to undergo egg retrieval steps again. Besides, it is also more economic for the families and health care organizations. Therefore, reproductive scientists have particular interests in improvement of ART outcomes in FET cycles. Previous investigations indicated the beneficial effects of fragment removal on improvement of embryo quality [17,18], blastocyst development [4], and clinical outcomes [19]. However, these studies are limited only to the fresh ART cycles. In addition, the fine structure of fragments in vitrified-warmed cleavage stage embryos is poorly understood. Currently, it is not clear whether vitrification and warming process alters the fine structure of fragments which might affect the success of FET cycles. Therefore, the present prospective randomized study, for the first time, investigated the effects of cosmetic micromanipulation of the cleavage stage embryos on ART outcomes in FET cycles. In addition, the potential link between ultrastructural changes of the fragments in vitrified-warmed cleavage embryos is assessed with its subsequent effects on ART. 2. Materials and methods 2.1. Patients This prospective randomized study was approved by ethic committee and institutional review board in June 2014. A cohort of 96 patients with similar clinical characteristics that underwent FET at Research and Clinical Center for Infertility, Yazd, Iran, from August 2014 to November 2015 was recruited to the study. A signed written consent form was obtained from each patient. The patients had history of previously undergone IVF or intra-cytoplasmic sperm injection (ICSI) with embryo cryopreservation. The study was performed only in patients that had days 2 or 3 vitrifiedwarmed embryos at 4–8 cells stages with grades of B or C. The patients were randomized into three groups of CM (n = 32), sham (n = 32) and control (n = 32). Block randomization was used for random allocation of patients to the study arms, with block size of six. The total numbers of needed blocks for study were 16 and the authors selected these blocks from all 20 possible blocks that created by combination of three by six. The selection of blocks was conducted by simple random method. In the CM group, the vitrified-warmed embryos were subjected to laser assisted zona hatching (LAH) prior to cosmetic micromanipulation. In the sham group, the vitrified-warmed embryos were only subjected to LAH; while, no intervention was taken for the control group. This trial was registered at www.irct.ir (IRCT2015030421332N1). The controlled ovarian hyperstimulation (COH), gamete handling and in vitro fertilization procedures was performed according to the protocols described previously [20]. 2.2. Vitrification and warming procedures For vitrification, the embryos were vitrified according to protocol that was described by Safari et al. previously [21]. In brief, the embryos were transferred to an equilibration solution
containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO) for 5–10 min (depending on the time necessary for cell re-expansion) and to the vitrification solution containing 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/L sucrose for 1 min. Then, the embryos were placed on the polyester sheet of Cryotop (Kitazato Co., Fujinomiya, Japan) with a minimum volume of vitrification solution. The sheets were quickly immersed into liquid nitrogen and capped, then transferred to a storage container. All vitrification stages were performed at room temperature. For warming, after pulling up from liquid nitrogen, the sheets were immersed directly into warming solution containing 1 mol/L sucrose and remained for 1 min at 37 C. Afterwards, the embryos were sequentially placed in diluents solution that contained of 0.5 mol/L sucrose and 0.25 mol/L sucrose, respectively and remained in each of these solutions for 3 min. After several washes, the embryos were placed in droplets of G-1TMv5 medium under oil. 2.3. Evaluation of vitrified-warmed embryo morphology After warming, the morphological parameters of embryos, including appearance and evenness of blastomeres, grade, degree and pattern of fragmentation were evaluated using 400 magnification of an inverted microscope (Nikon TE 300, Japan). The vitrified-warmed embryos were morphologically scored according to Hill et al. [22]: Grades A: Even blastomeres with no fragments. B: A little inequality in size of blastomeres, <10% cytoplasmic fragments. C: Unequal sized blastomeres with <50% fragmentation. D: Unequal blastomeres with severe cytoplasmic fragmentation and large black granules. The fragmentation degree was expressed as a percentage and was defined as a volume of the PVS and/or cleavage cavity occupied by fragments. According to Alikani et al. [19], the embryos were classified based on distribution and size of the fragments to five patterns. In type 1, fragments are minimal in volume, typically associated with only one blastomere. Fragments in type II are localized and mostly occupy the PVS. In type III, fragments are small and scattered that may be positioned in the cleavage cavity or periphery. Fragments in type IV are large, sometimes resembling whole blastomeres. These fragments are randomly distributed and associated with uneven blastomeres. In type V, fragmentation appears the process of necrosis and the cytoplasm of blastomeres is very granular. Multinucleated embryos were excluded from this study. After embryo assessment, the embryos in three groups were cultured in an environment of 5% O2, 6% CO2 at 37 C for overnight until ET. 2.4. Cosmetic micromanipulation The LAH procedure was performed for all embryos in CM group by using a 1480 nm infrared diode laser beam to open 13–15 mm hole in the zona pellucida (ZP). Cosmetic micromanipulation procedure was performed 4–5 h after embryo warming and incubation, on the heated stage of a Nikon inverted microscope (600 magnification), using Eppendorf hydraulic micromanipulators (Eppendorf, France). A micropipette with 13–15 mm inner diameter was used for the procedure. All the embryos in CM group were subjected to fragment removal. while, in embryos with coarse granulation in addition to fragments, the coarse granules were removed as much as possible. Continuous refocusing and rotation of the embryos were performed to avoid damaging the neighboring intact blastomeres and to provide access to more fragments and coarse granules. In embryos with coarse granulation, the micropipette was gently moved closed to debris and granules in PVS to aspirate them into the micropipette. After micromanipulation and morphology evaluation, the embryos were washed several times in fresh medium and cultured at 5% O2, 6% CO2 with 37 C until ET.
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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2.5. Embryo transfer and endometrial preparation
2.7. Transmission electron microscopy (TEM)
From the second day of menstrual cycle, the patients received estradiol valerate (Aburaihan Co, Tehran, Iran) orally at the dose of 6 mg per day until endometrial thickness reached 8 mm and triple line in ultrasonography. At this time, 100 mg progesterone in oil (Aburaihan, Co, Tehran, Iran) was injected daily. Warming of the day 2 and day 3 embryos was performed on days 2 and 3 after beginning of progesterone injection, respectively. ET was performed overnight after cosmetic micromanipulation and culture of vitrified-warmed embryos. At the same time, the quality of the embryos was re-evaluated using inverted microscope. Evenness of blastomeres, fragmentation degree, embryo grading and presence of lysed blastomeres were recorded. Only dividing embryos in all groups were subjected to ET. Estradiol and progesterone administration were continued until the 10th gestational week.
The fragmented vitrified-warmed embryo was fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) solution and rinsed in PBS several times. The sample was post-fixed with 1% osmium tetroxide in PBS, dehydrated using an ascending series of ethanol, and embedded in resin. Leica EM UC6 Ultramicrotome (Leica Microsystems Inc., Germany) was used for ultrathin sectioning. The semi-thin sections of 500 nm were stained with toluidine blue and examined by light microscope to check the samples. Ultra-thin sections (70 nm) were cut using a diamond knife, mounted on copper grids and stained with uranyl acetate followed by lead citrate. The ultrathin sections were examined by TEM (Zeiss, Germany).
2.6. Main outcome measures Outcome measures were done as follows, chemical pregnancy was confirmed by measuring bhCG level 14 days post FET. Clinical pregnancy was confirmed with presence of fetal heartbeat that was evaluated in the 7th gestational week by ultrasonography. Implantation rate was reported by total number of intrauterine gestational sacs divided by total number of transferred embryos multiplied by 100. Ongoing pregnancy was defined as pregnancy continuation beyond the 12th week of gestation. Stillbirth was defined as death of a child after 20 weeks of gestational age in intrauterine or intrapartum. Delivery rate was recorded as a ratio between deliveries and ET cycles, and live birth rate as a ratio between umber of healthy newborns and number of ET cycles.
2.8. Statistical analysis Distribution and normality of data was analyzed by Kolmogorov–Smirnov test and accordingly, for continuous variables which were normally distributed, ANOVA test and for data that were not normally distributed, non-parametric Kruscall-Walis test were applied. Indeed, for analysis of categorical variables, Chi-square test was used. P value <0.05 was considered statistically significant. Data analysis was performed with SPSS 20 software. Four patients from two groups of CM and sham were excluded from analysis due to lost to follow up. 3. Results 3.1. Patients characteristics The patients’ clinical and laboratory characteristics in the last fresh cycles in three groups of CM, sham and control are presented in Table 1. There were no significant differences between the three
Table 1 Comparison of clinical and laboratory characteristics in the last fresh cycles amongst the groups. Parameters
Groups
P value
CM (n = 30)
Sham (n = 30)
Control (n = 32)
Type of infertility (%) Primary Secondary
58.6 41.4
63.3 36.7
62.5 37.5
0.92
Cause of infertility (%) Female factor Male factor Both Unexplained
43.3 40.0 13.3 3.3
50.0 30.0 20.0 0.0
43.8 28.1 28.1 0.0
0.59
COH protocol (%) Agonist Antagonist
20.0 80.0
23.3 76.7
18.8 81.3
0.90
Peaked Estradiol (pg/ml) *
2682.90 1793.40
2719.95 1641.21
2470.44 1499.84
0.89
Based FSH, mIU/ml*
5.95 2.63
6.43 2.34
5.55 2.14
0.39
ART type (%) IVF ICSI IVF/ICSI
36.7 56.7 6.7
33.3 56.7 10.0
28.1 62.5 9.4
0.95
No. of retrieved oocytes*
21.57 11.14
18.10 8.19
19.75 12.54
0.46
No. of MII oocytes*
19.53 11.02
16.79 7.59
18.25 11.72
0.59
No. of fertilized oocytes**
8.5 (4–36)
10 (3–25)
9.5 (1–36)
0.73
No. of cleaved embryos**
7 (4–30)
9 (3–24)
9 (1–30)
0.66
*Values are mean SD or **median (minimum-maximum)
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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group (P = 0.00001). After overnight culturing, the embryos that were treated with cosmetic micromanipulation showed dramatic persistence in diminishing fragmentation until ET (3.30 3.19) (Fig. 1), when compared to the sham (17.32 11.30) or to the control groups (19.81 11.80). In regards to blastomeres evenness; over 60% of the embryos in three groups did not show evenness in blastomeres size at the time of warming. Interestingly, the embryos treated with cosmetic micromanipulation adopted significantly higher blostomeres evenness than those in the sham or the control groups (65.7% vs 39.4% and 34.2%; respectively) after overnight incubation. After warming, the embryo grades in all groups was similar; as over 70% of the embryos in all groups characterized as C grade. However, post cosmetic micromanipulation and subsequent overnight incubation, significant improvement in embryo grade was observed compared to the sham and control groups (P = 0.00001). So that grade C embryos were reduced from 83% to 4.3% and grades A and B embryos were increased from 0% and 17% to 34.3% and 61.4%, respectively (P = 0.00001). In contrary, sham and control groups remained with around 70% of grade C and the rest had no embryos in grade A.
groups in regards to type and cause of infertility, protocols for COH, the peak level of estradiol, FSH level in day 3, number of IVF or ICSI cycles, number of retrieved MII and fertilized oocytes and number of cleaved embryos. The patients’ characteristics in present FET cycles are summarized in Table 2. Patients’ age, duration of infertility, history of previous failed implantation and number of transferred embryos were similar between all groups. 3.2. Embryo morphology post warming and culturing For assessment of the impact of cosmetic micromanipulation on embryo morphology, we evaluated the degree of fragmentation, blastomeres evenness and embryo grades immediately after embryo warming, overnight after cosmetic micromanipulation and before ET (Table 3). The embryos after warming in regards to cell appearance, pattern of fragmentation and rate of fragmented embryos with course granulation were similar in the all groups. The means of fragmentation rates after warming in CM (17.71 10.55), sham (15.35 10.19) and control (17.47 10.97) groups were comparable. However, immediately after cosmetic micromanipulation, it was significantly reduced to 2.5% in CM Table 2 Comparison of patients’ characteristics in present FET cycle amongst the groups. Parameters
Groups
Female age (mean SD) Duration of infertility(years)a History of previous failed implantation (%) No. of transferred embryosa a
P value
CM (n = 30)
Sham (n = 30)
Control (n = 32)
29.73 4.60 4 (2–20) 66.7% 2 (1–3)
30.60 5.60 5.5 (2–20) 56.7% 2 (1–3)
29.23 5.26 5 (1–16) 65.6% 2 (1–3)
0.58 0.89 0.67 0.78
Values are median (minimum–maximum).
Table 3 Comparison of morphological characteristics of embryos immediately after warming and after overnight culture amongst the groups Parameters
Groups
P value
CM (n = 70)
Sham (n = 71)
Control (n = 79)
Percent fragmentation after warming*
17.71 10.55a
15.35 10.19
17.47 10.97
0.34
Percent fragmentation immediately after cosmetic micromanipulation*
2.40 2.65b
–
–
–
17.32 11.30
19.81 11.80
0.00001
c
***
Percent fragmentation after overnight culture*
3.30 3.19
Evenness after warming (%)
31.4
38.0
38.5
0.61
Evenness after overnight culture (%)
65.7
39.4
34.2
0.00001
Pattern of fragmentation after warming (%) I II III IV
24.3 22.9 42.9 10
26.8 18.3 45.1 9.9
20.3 20.3 43.0 16.5
0.83
Embryo grade after warming (%) A B C
0.0 17.1 82.9
0.0 31.0 69.0
0.0 22.8 77.2
0.15
Embryo grade after overnight culture (%) A B C
34.3 61.4 4.3
0.0 31.0 69.0
0.0 17.7 82.3
0.00001***
Fragmented embryos with course granulation (%)
7.1
11.3
5.1
0.35
Normal cell appearance (%)
80.0
85.9
84.8
0.6
Significantly different a vs. b, a vs. c (P = 0.00001), *Values are mean SD. *** Statics is significant at 0.05.
b,c
***
Not significantly different b vs. c (P > 0.05).
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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Fig. 1. Beneficial effect of cosmetic micromanipulation on subsequent development of fragmented vitrified-warmed embryos. (a) fragmented embryos before cosmetic micromanipulation, (b) the same embryos immediately after cosmetic micromanipulation and (c) after overnight culture and immediately before ET. 1a, 2a are day 3 and 3a, 4a are day 2 vitrified warmed embryos. The embryo in 1c depicts the initiation of compaction. Embryos in 1a, 2a and 3a only subjected to fragment removal; the embryo in 4a subjected to both fragment removal and to debris or coarse granules removal. Magnification, 400.
3.3. Clinical outcomes The rates of clinical and ongoing pregnancies, implantation, delivery and live birth as well as child genders were all similar in the three groups (Table 4). Neonatal death was not observed in any of the studied groups. In each group, only one of pregnancies was twin and the rest were singleton. One twin pregnancy in control group was led to still birth in 6th month of gestation. Whereas, still births were not observed in CM and sham groups. In addition, coarse granulation was observed in a small number of fragmented embryos in CM, sham and control groups. Transferring of these embryos did not lead to any pregnancy. 3.4. Ultrastructure of cytoplasmic fragments The general architecture of embryo fragments was reasonably well preserved (Fig. 2). The main features of cryoinjury, such as plasma membrane disruption, extensive disorganization of the cytoplasm and vacuolization were not observed in these
fragments. The fragments were much smaller than the blastomeres and contained the same complement of normal appearing organelles, when compared with the adjacent blastomeres. In addition, intact and distinct membranes with some remnants of microvilli were observed in almost all of the fragments. Around the fragments, small blebs of cytoplasm and cellular debris were detected. The smooth endoplasmic reticulum (SER) for the most part was vesicular, containing low electron-dense materials. In addition, considerable number of mitochondria were distributed in fragments in comparison to the other cytoplasmic organelles. The majority of mitochondria were intact, rounded to oval in shape and possessed few peripheral cristae with a moderately electron-dense matrix. They were either isolated or associated with SER vesicles forming mitochondria-vesicle (MV) complexes. MV complexes appeared as SER vesicles filled with flocculent and slightly electron-dense material surrounded by mitochondria. The fragments occasionally had remnant of intact undischarged electrondense cortical granules (CGs) beneath the plasma membrane. Annulate lamellae (AL) were occasionally seen and consisted of
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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Table 4 Comparison of clinical outcomes amongst the groups. Variables
Groups CM (n = 30)
Sham (n = 30)
Control (n = 32)
Chemical pregnancy Clinical pregnancy Implantation rate Ongoing pregnancy Delivery rate Live birth rate gender ratio Male Female
8/30 5/30 9/70 5/30 5/30 6/30
8/30 7/30 9/71 7/30 7/30 8/30
12/32 (37.5) 11/32 (34.4) 12/79 (15.2) 11/32 (34.4) 10/32 (31.3) 10/32 (31.3)
0.36 0.19 0.88 0.19 0.40 0.43
5 (50.0) 5 (50.0)
0.41
(26.7) (16.7) (12.9) (16.7) (16.7) (20)
5 (83.3) 1 (16.7)
P value
(26.7) (23.3) (12.7) (23.3) (23.3) (26.7)
5 (62.5) 3 (37.5)
Values are number/total (%).
parallel stacks of cisternae. In addition, remnants of regressing mitochondria, membranes belonging to the Golgi complexes, small clear vacuoles, primary and secondary lysosomes were occasionally found within the fragments. Primary lysosomes were small with dense cores. 4. Discussion The precise etiology of embryo fragmentation is unknown. Various hypotheses have been proposed to explain fragment formation, including an instability within the cortical microfilament network [23], chromosomal abnormalities such as mosaicism and aneuploidy [24] and apoptotic and necrotic processes [25]. In this regard, fragment formation may not have a common origin [23]. In spite of the unknown mechanism underlying the generation of fragments, it is clear that this phenomenon is associated with deleterious ART outcomes [4–8]. Therefore, removal of the embryo fragments have been suggested for enhancing the ART outcomes [19]. Following introducing of embryo fragment removal from 1990s until now, the beneficial effect of fragment removal on low-grade embryos remained as a controversial issue. Only limited retrospective studies have been done in this era that suggested improvement of implantation and clinical pregnancy rates [13,19]. On the other hand, the recent prospective studies did not show any significant improvement in the ART outcomes [18,26,27]. These studies were, however, done on fresh ET cycles and the effect of fragment removal on vitrified-warmed embryos is not known yet. Our prospective randomized study, for the first time, investigated the effects of cosmetic micromanipulation of the cleavage stage embryos on ART outcomes in FET cycles. Also, the potential link between ultrastructural changes of embryo fragments post vitrification was assessed with subsequent effects on ART. The TEM data showed that fragment components including organelles, cytoplasm texture and membrane in vitrified-warmed embryo was well preserved and comparable to the fragments in fresh cleavage embryos [28–30]. In spite of the notion that lysing fragments release toxic materials that can damage the neighboring blastomeres [31], there were no signs of degeneration or lysis due to cryoinjury in our study. Therefore, we conclude that vitrification and warming method did not cause adverse effects on fragments fine structure. In addition, the majority of blastomere’s organelles, except the nucleus, were present in the fragments. This finding supports the opinion that large fragments result in the loss of large volumes of blastomere’s cytoplasm, and deplete the blastomeres from the essential organelles and vital materials, such as mitochondria, mRNA and proteins that can lead to developmental arrest in embryo [32]. Similar to Halvaei’s observation in fresh embryos [26], intact and distinct membrane with some remnants of microvilli were noticed in fragments. In addition, fragments exhibited a uniform cytoplasmic texture with considerable
Fig. 2. Ultrastructure of cytoplasmic fragments in vitrified-warmed embryo by TEM. B, blastomere; M, mitochondria; rM, remnant of regressing mitochondrion; V, SER vesicles; MV, mitochondria-vesicle complex; Ly, primary lysosome; G, membranes belonging to Golgi complexes; mv, microvilli; Va, vacuoles; AL, annulate lamella; CG, cortical granules.
number of mitochondria in comparison to the other cytoplasmic organelles. Our data also indicated that after overnight culturing, the embryos that were subjected to cosmetic micromanipulation, had significantly persistent diminished fragmentation when compared to either sham or control groups. In the CM group, over 90% of the FETs had only 0–5% embryo fragmentations; and in total, none of
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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the ETs in this group involved with embryos >10% fragmentations. The morphological grades improvement was maintained after overnight until ET, which is in agreement with studies on fresh ET cycles [17,18]. The evenness embryos morphology following overnight culturing after cosmetic micromanipulation suggests that the removal of fragments from cleavage cavity and around the blastomeres might modulate the blastomeres division and improve the embryo evenness as hypothesized by Alikani et al. [19]. The fragments positioned in the cleavage cavity may lead to distortion of the division planes or interfere with normal cell–cell contact [19]. In spite of the significant improvements in quality and grade of the embryos after cosmetic micromanipulation and before ET, the clinical outcomes (e.g., implantation, multiple pregnancies, delivery and live birth rates) were not different from the sham and control groups. These findings are in line with the previous prospective investigations on fragment removal in fresh ET cycles [18,27]. In addition, Halvaei and coworkers showed no improvement on the ART clinical outcomes after performing cosmetic micromanipulation on embryos in fresh ET cycles [26]. Besides, coarse granulation was observed in a small number of fragmented embryos in CM and other groups. Transferring these embryos did not lead to any pregnancy in all studied groups, even after granulation removal. The results are also supported by the study of Farhi et al. that showed ET of embryos resulting from oocytes with coarse granulation in their PVS had negative effects on ART outcomes [14]. Lacks of noticeable success in clinical outcomes of transferred embryos post fragment and/or coarse granulation removals in our study and others might be explained by the fact that all of the studies in this era were done on small cohorts [18,26,27], which did not provide strong statistical power to show the exact role of fragment removal in ART outcomes. It also may be linked to several mechanisms that impair or eliminate embryo developmental viability, such as chromosomal abnormalities [33–36], loss of regulatory proteins localized in polarized domains due to fragmentation [23] and loss of essential part of cytoplasm which carries cytoplasmic organelles with fragments [32]. These aforementioned variables are possible reasons for futile effect of fragment removal on clinical outcomes. Also, it was described that embryo development depends on the energy (ATP) production by the mitochondria. Therefore, losing these organelles by fragmentation or exposure of embryo for extended time out of the incubator during fragment removal, may interfere with the viability and implantation potential of the embryos [26]. In conclusion, embryo vitrification had no adverse effects on fine structure of the fragments. In addition, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters, but no noticeable improvements were noticed in ART clinical outcomes. Conflict of interest The authors declare that they have no conflict of interest. Author’s roles SS and IH collected and analyzed the data. SS and SAN interpreted the TEM micrographs. MAK and MA involved in the study design. SS and ZB drafted the article, and all authors critically revised it for important intellectual content. All authors approved the final version of the manuscript. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Acknowledgments This study was extracted from the Ph.D. thesis of Somayyeh Safari. The authors gratefully thank Research and Clinical Center for Infertility, Yazd, Iran, for providing all supports.
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[26] Halvaei I, Khalili MA, Esfandiari N, Safari S, Talebi AR, Miglietta S, et al. Ultrastructure of cytoplasmic fragments in human cleavage stage embryos. J Assist Reprod Genet 2016. [27] E. Sozen, K. Vicdan, C. Akarsu, G. Tuncay, B. Buluc, M. Colak, A prospective, randomized, controlled study of fragment removal in women who have moderate fragmentated embryos, Human Reproduction, Oxford Univ Press Great Clarendon St, Oxford Ox2 6dp, England; 2012. [28] Trounson A, Sathananthan A. The application of electron microscopy in the evaluation of two-to four-cell human embryos cultured in vitro for embryo transfer. J In Vitro Fertil Embryo Transf 1984;1:153–65. [29] Sathananthan AH, Wood C, Leeton JF. Ultrastructural evaluation of 8–16 cell human embryos cultured in vitro. Micron 1969;1982(13):193–203. [30] Sathananthan H, Bongso A, Ng S-C, Ho J, Mok H, Ratnam S. Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells. Hum Reprod 1990;5:309–18.
[31] Chi H-J, Koo J-J, Choi S-Y, Jeong H-J, Roh S-I. Fragmentation of embryos is associated with both necrosis and apoptosis. Fertil Steril 2011;96:187–92. [32] Johansson M, Hardarson T, Lundin K. There is a cutoff limit in diameter between a blastomere and a small anucleate fragment. J Assist Reprod Genet 2003;20:309–13. [33] Bongso A, Ng S-C, Lim J, Fong C-Y, Ratnam S. Preimplantation genetics: chromosomes of fragmented human embryos. Fertil Steril 1991;56:66–70. [34] Munné S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382–91. [35] Magli M, Gianaroli L, Ferraretti A. Chromosomal abnormalities in embryos. Mol Cell Endocrinol 2001;183:S29–34. [36] Racowsky C, Ohno-Machado L, Kim J, Biggers JD. Is there an advantage in scoring early embryos on more than one day. Hum Reprod 2009;24:2104–13.
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Original article
Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments Somayyeh Safaria , Mohammad Ali Khalilia,* , Zeinab Barekatib , Iman Halvaeic , Morteza Anvaria , Stefania A. Nottolad a
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Genome Plasticity, Department of Biomedicine, University of Basel, Switzerland c Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran d Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University of Rome, Rome, Italy b
A R T I C L E I N F O
Article history: Received 19 February 2017 Received in revised form 6 April 2017 Accepted 6 May 2017 Available online xxx Keywords: Cosmetic micromanipulation Fragment removal Vitrified-warmed embryos Pregnancy outcome Transmission electron microscopy
A B S T R A C T
The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n = 32), sham (n = 32) and control (n = 32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P = 0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrifiedwarmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments. © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
1. Introduction Fragmentation or generation of small anuclear cytoplasmic fragments is a common feature of human developing embryos [1,2]. In various embryo grading systems, the degree of fragmentation and the developmental stage of embryos are defined as the most common used criteria for selecting a high-quality embryo for
* Corresponding author at: Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Bouali Ave, Safaeiyeh, Yazd, 89168-77391, Iran. E-mail addresses: [email protected] (S. Safari), [email protected] (M.A. Khalili), [email protected] (Z. Barekati), [email protected] (I. Halvaei), [email protected] (M. Anvari), [email protected] (S.A. Nottola).
transfer following in vitro fertilization (IVF) [3]. In spite of the unknown mechanism underlying the generation of fragments, it is clear that this phenomenon is associated with a deleterious assisted reproductive technology (ART) outcomes [4–8]. For example, further development of fragmented embryos is impaired, often followed by cleavage arrest and degeneration. In addition, transfer of these embryos showed limited developmental potential and rarely resulted in successful pregnancy [5,9]. Therefore, fragmentation is an important biomarker for poor pregnancy outcomes [1,10–12]. It has been proposed that cytoplasmic fragmentation induces apoptosis and limits blastomeres cleavage rate. Therefore, removal of these fragments may improve cell division and implantation [13]. In addition to fragments formation in embryos, the perivitelline space (PVS) in fragmented embryos may be filled with other debris,
http://dx.doi.org/10.1016/j.repbio.2017.05.003 1642-431X/© 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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such as coarse granules. Farhi and co-workers showed that this dysmorphism had a negative impact on ART outcome, and transfer of embryos generated from oocytes with PVS coarse granulation resulted in significant lower implantation and pregnancy rates [14]. Recently Halvaei et al. showed that removal of fragments and coarse granules (cosmetic micromanipulation) from low-grade embryos improved pregnancy rates in patients with implantation failure in fresh cycles [15]. Frozen embryo transfer (FET) cycles constitute approximately 20% of all performed ART cycles in assisted reproduction clinics [16]. It is important to note that using frozen embryos in ART cycles is less invasive for the mother rather than starting a new cycle of hormone therapy to stimulate ovaries and to undergo egg retrieval steps again. Besides, it is also more economic for the families and health care organizations. Therefore, reproductive scientists have particular interests in improvement of ART outcomes in FET cycles. Previous investigations indicated the beneficial effects of fragment removal on improvement of embryo quality [17,18], blastocyst development [4], and clinical outcomes [19]. However, these studies are limited only to the fresh ART cycles. In addition, the fine structure of fragments in vitrified-warmed cleavage stage embryos is poorly understood. Currently, it is not clear whether vitrification and warming process alters the fine structure of fragments which might affect the success of FET cycles. Therefore, the present prospective randomized study, for the first time, investigated the effects of cosmetic micromanipulation of the cleavage stage embryos on ART outcomes in FET cycles. In addition, the potential link between ultrastructural changes of the fragments in vitrified-warmed cleavage embryos is assessed with its subsequent effects on ART. 2. Materials and methods 2.1. Patients This prospective randomized study was approved by ethic committee and institutional review board in June 2014. A cohort of 96 patients with similar clinical characteristics that underwent FET at Research and Clinical Center for Infertility, Yazd, Iran, from August 2014 to November 2015 was recruited to the study. A signed written consent form was obtained from each patient. The patients had history of previously undergone IVF or intra-cytoplasmic sperm injection (ICSI) with embryo cryopreservation. The study was performed only in patients that had days 2 or 3 vitrifiedwarmed embryos at 4–8 cells stages with grades of B or C. The patients were randomized into three groups of CM (n = 32), sham (n = 32) and control (n = 32). Block randomization was used for random allocation of patients to the study arms, with block size of six. The total numbers of needed blocks for study were 16 and the authors selected these blocks from all 20 possible blocks that created by combination of three by six. The selection of blocks was conducted by simple random method. In the CM group, the vitrified-warmed embryos were subjected to laser assisted zona hatching (LAH) prior to cosmetic micromanipulation. In the sham group, the vitrified-warmed embryos were only subjected to LAH; while, no intervention was taken for the control group. This trial was registered at www.irct.ir (IRCT2015030421332N1). The controlled ovarian hyperstimulation (COH), gamete handling and in vitro fertilization procedures was performed according to the protocols described previously [20]. 2.2. Vitrification and warming procedures For vitrification, the embryos were vitrified according to protocol that was described by Safari et al. previously [21]. In brief, the embryos were transferred to an equilibration solution
containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO) for 5–10 min (depending on the time necessary for cell re-expansion) and to the vitrification solution containing 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/L sucrose for 1 min. Then, the embryos were placed on the polyester sheet of Cryotop (Kitazato Co., Fujinomiya, Japan) with a minimum volume of vitrification solution. The sheets were quickly immersed into liquid nitrogen and capped, then transferred to a storage container. All vitrification stages were performed at room temperature. For warming, after pulling up from liquid nitrogen, the sheets were immersed directly into warming solution containing 1 mol/L sucrose and remained for 1 min at 37 C. Afterwards, the embryos were sequentially placed in diluents solution that contained of 0.5 mol/L sucrose and 0.25 mol/L sucrose, respectively and remained in each of these solutions for 3 min. After several washes, the embryos were placed in droplets of G-1TMv5 medium under oil. 2.3. Evaluation of vitrified-warmed embryo morphology After warming, the morphological parameters of embryos, including appearance and evenness of blastomeres, grade, degree and pattern of fragmentation were evaluated using 400 magnification of an inverted microscope (Nikon TE 300, Japan). The vitrified-warmed embryos were morphologically scored according to Hill et al. [22]: Grades A: Even blastomeres with no fragments. B: A little inequality in size of blastomeres, <10% cytoplasmic fragments. C: Unequal sized blastomeres with <50% fragmentation. D: Unequal blastomeres with severe cytoplasmic fragmentation and large black granules. The fragmentation degree was expressed as a percentage and was defined as a volume of the PVS and/or cleavage cavity occupied by fragments. According to Alikani et al. [19], the embryos were classified based on distribution and size of the fragments to five patterns. In type 1, fragments are minimal in volume, typically associated with only one blastomere. Fragments in type II are localized and mostly occupy the PVS. In type III, fragments are small and scattered that may be positioned in the cleavage cavity or periphery. Fragments in type IV are large, sometimes resembling whole blastomeres. These fragments are randomly distributed and associated with uneven blastomeres. In type V, fragmentation appears the process of necrosis and the cytoplasm of blastomeres is very granular. Multinucleated embryos were excluded from this study. After embryo assessment, the embryos in three groups were cultured in an environment of 5% O2, 6% CO2 at 37 C for overnight until ET. 2.4. Cosmetic micromanipulation The LAH procedure was performed for all embryos in CM group by using a 1480 nm infrared diode laser beam to open 13–15 mm hole in the zona pellucida (ZP). Cosmetic micromanipulation procedure was performed 4–5 h after embryo warming and incubation, on the heated stage of a Nikon inverted microscope (600 magnification), using Eppendorf hydraulic micromanipulators (Eppendorf, France). A micropipette with 13–15 mm inner diameter was used for the procedure. All the embryos in CM group were subjected to fragment removal. while, in embryos with coarse granulation in addition to fragments, the coarse granules were removed as much as possible. Continuous refocusing and rotation of the embryos were performed to avoid damaging the neighboring intact blastomeres and to provide access to more fragments and coarse granules. In embryos with coarse granulation, the micropipette was gently moved closed to debris and granules in PVS to aspirate them into the micropipette. After micromanipulation and morphology evaluation, the embryos were washed several times in fresh medium and cultured at 5% O2, 6% CO2 with 37 C until ET.
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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2.5. Embryo transfer and endometrial preparation
2.7. Transmission electron microscopy (TEM)
From the second day of menstrual cycle, the patients received estradiol valerate (Aburaihan Co, Tehran, Iran) orally at the dose of 6 mg per day until endometrial thickness reached 8 mm and triple line in ultrasonography. At this time, 100 mg progesterone in oil (Aburaihan, Co, Tehran, Iran) was injected daily. Warming of the day 2 and day 3 embryos was performed on days 2 and 3 after beginning of progesterone injection, respectively. ET was performed overnight after cosmetic micromanipulation and culture of vitrified-warmed embryos. At the same time, the quality of the embryos was re-evaluated using inverted microscope. Evenness of blastomeres, fragmentation degree, embryo grading and presence of lysed blastomeres were recorded. Only dividing embryos in all groups were subjected to ET. Estradiol and progesterone administration were continued until the 10th gestational week.
The fragmented vitrified-warmed embryo was fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) solution and rinsed in PBS several times. The sample was post-fixed with 1% osmium tetroxide in PBS, dehydrated using an ascending series of ethanol, and embedded in resin. Leica EM UC6 Ultramicrotome (Leica Microsystems Inc., Germany) was used for ultrathin sectioning. The semi-thin sections of 500 nm were stained with toluidine blue and examined by light microscope to check the samples. Ultra-thin sections (70 nm) were cut using a diamond knife, mounted on copper grids and stained with uranyl acetate followed by lead citrate. The ultrathin sections were examined by TEM (Zeiss, Germany).
2.6. Main outcome measures Outcome measures were done as follows, chemical pregnancy was confirmed by measuring bhCG level 14 days post FET. Clinical pregnancy was confirmed with presence of fetal heartbeat that was evaluated in the 7th gestational week by ultrasonography. Implantation rate was reported by total number of intrauterine gestational sacs divided by total number of transferred embryos multiplied by 100. Ongoing pregnancy was defined as pregnancy continuation beyond the 12th week of gestation. Stillbirth was defined as death of a child after 20 weeks of gestational age in intrauterine or intrapartum. Delivery rate was recorded as a ratio between deliveries and ET cycles, and live birth rate as a ratio between umber of healthy newborns and number of ET cycles.
2.8. Statistical analysis Distribution and normality of data was analyzed by Kolmogorov–Smirnov test and accordingly, for continuous variables which were normally distributed, ANOVA test and for data that were not normally distributed, non-parametric Kruscall-Walis test were applied. Indeed, for analysis of categorical variables, Chi-square test was used. P value <0.05 was considered statistically significant. Data analysis was performed with SPSS 20 software. Four patients from two groups of CM and sham were excluded from analysis due to lost to follow up. 3. Results 3.1. Patients characteristics The patients’ clinical and laboratory characteristics in the last fresh cycles in three groups of CM, sham and control are presented in Table 1. There were no significant differences between the three
Table 1 Comparison of clinical and laboratory characteristics in the last fresh cycles amongst the groups. Parameters
Groups
P value
CM (n = 30)
Sham (n = 30)
Control (n = 32)
Type of infertility (%) Primary Secondary
58.6 41.4
63.3 36.7
62.5 37.5
0.92
Cause of infertility (%) Female factor Male factor Both Unexplained
43.3 40.0 13.3 3.3
50.0 30.0 20.0 0.0
43.8 28.1 28.1 0.0
0.59
COH protocol (%) Agonist Antagonist
20.0 80.0
23.3 76.7
18.8 81.3
0.90
Peaked Estradiol (pg/ml) *
2682.90 1793.40
2719.95 1641.21
2470.44 1499.84
0.89
Based FSH, mIU/ml*
5.95 2.63
6.43 2.34
5.55 2.14
0.39
ART type (%) IVF ICSI IVF/ICSI
36.7 56.7 6.7
33.3 56.7 10.0
28.1 62.5 9.4
0.95
No. of retrieved oocytes*
21.57 11.14
18.10 8.19
19.75 12.54
0.46
No. of MII oocytes*
19.53 11.02
16.79 7.59
18.25 11.72
0.59
No. of fertilized oocytes**
8.5 (4–36)
10 (3–25)
9.5 (1–36)
0.73
No. of cleaved embryos**
7 (4–30)
9 (3–24)
9 (1–30)
0.66
*Values are mean SD or **median (minimum-maximum)
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group (P = 0.00001). After overnight culturing, the embryos that were treated with cosmetic micromanipulation showed dramatic persistence in diminishing fragmentation until ET (3.30 3.19) (Fig. 1), when compared to the sham (17.32 11.30) or to the control groups (19.81 11.80). In regards to blastomeres evenness; over 60% of the embryos in three groups did not show evenness in blastomeres size at the time of warming. Interestingly, the embryos treated with cosmetic micromanipulation adopted significantly higher blostomeres evenness than those in the sham or the control groups (65.7% vs 39.4% and 34.2%; respectively) after overnight incubation. After warming, the embryo grades in all groups was similar; as over 70% of the embryos in all groups characterized as C grade. However, post cosmetic micromanipulation and subsequent overnight incubation, significant improvement in embryo grade was observed compared to the sham and control groups (P = 0.00001). So that grade C embryos were reduced from 83% to 4.3% and grades A and B embryos were increased from 0% and 17% to 34.3% and 61.4%, respectively (P = 0.00001). In contrary, sham and control groups remained with around 70% of grade C and the rest had no embryos in grade A.
groups in regards to type and cause of infertility, protocols for COH, the peak level of estradiol, FSH level in day 3, number of IVF or ICSI cycles, number of retrieved MII and fertilized oocytes and number of cleaved embryos. The patients’ characteristics in present FET cycles are summarized in Table 2. Patients’ age, duration of infertility, history of previous failed implantation and number of transferred embryos were similar between all groups. 3.2. Embryo morphology post warming and culturing For assessment of the impact of cosmetic micromanipulation on embryo morphology, we evaluated the degree of fragmentation, blastomeres evenness and embryo grades immediately after embryo warming, overnight after cosmetic micromanipulation and before ET (Table 3). The embryos after warming in regards to cell appearance, pattern of fragmentation and rate of fragmented embryos with course granulation were similar in the all groups. The means of fragmentation rates after warming in CM (17.71 10.55), sham (15.35 10.19) and control (17.47 10.97) groups were comparable. However, immediately after cosmetic micromanipulation, it was significantly reduced to 2.5% in CM Table 2 Comparison of patients’ characteristics in present FET cycle amongst the groups. Parameters
Groups
Female age (mean SD) Duration of infertility(years)a History of previous failed implantation (%) No. of transferred embryosa a
P value
CM (n = 30)
Sham (n = 30)
Control (n = 32)
29.73 4.60 4 (2–20) 66.7% 2 (1–3)
30.60 5.60 5.5 (2–20) 56.7% 2 (1–3)
29.23 5.26 5 (1–16) 65.6% 2 (1–3)
0.58 0.89 0.67 0.78
Values are median (minimum–maximum).
Table 3 Comparison of morphological characteristics of embryos immediately after warming and after overnight culture amongst the groups Parameters
Groups
P value
CM (n = 70)
Sham (n = 71)
Control (n = 79)
Percent fragmentation after warming*
17.71 10.55a
15.35 10.19
17.47 10.97
0.34
Percent fragmentation immediately after cosmetic micromanipulation*
2.40 2.65b
–
–
–
17.32 11.30
19.81 11.80
0.00001
c
***
Percent fragmentation after overnight culture*
3.30 3.19
Evenness after warming (%)
31.4
38.0
38.5
0.61
Evenness after overnight culture (%)
65.7
39.4
34.2
0.00001
Pattern of fragmentation after warming (%) I II III IV
24.3 22.9 42.9 10
26.8 18.3 45.1 9.9
20.3 20.3 43.0 16.5
0.83
Embryo grade after warming (%) A B C
0.0 17.1 82.9
0.0 31.0 69.0
0.0 22.8 77.2
0.15
Embryo grade after overnight culture (%) A B C
34.3 61.4 4.3
0.0 31.0 69.0
0.0 17.7 82.3
0.00001***
Fragmented embryos with course granulation (%)
7.1
11.3
5.1
0.35
Normal cell appearance (%)
80.0
85.9
84.8
0.6
Significantly different a vs. b, a vs. c (P = 0.00001), *Values are mean SD. *** Statics is significant at 0.05.
b,c
***
Not significantly different b vs. c (P > 0.05).
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Fig. 1. Beneficial effect of cosmetic micromanipulation on subsequent development of fragmented vitrified-warmed embryos. (a) fragmented embryos before cosmetic micromanipulation, (b) the same embryos immediately after cosmetic micromanipulation and (c) after overnight culture and immediately before ET. 1a, 2a are day 3 and 3a, 4a are day 2 vitrified warmed embryos. The embryo in 1c depicts the initiation of compaction. Embryos in 1a, 2a and 3a only subjected to fragment removal; the embryo in 4a subjected to both fragment removal and to debris or coarse granules removal. Magnification, 400.
3.3. Clinical outcomes The rates of clinical and ongoing pregnancies, implantation, delivery and live birth as well as child genders were all similar in the three groups (Table 4). Neonatal death was not observed in any of the studied groups. In each group, only one of pregnancies was twin and the rest were singleton. One twin pregnancy in control group was led to still birth in 6th month of gestation. Whereas, still births were not observed in CM and sham groups. In addition, coarse granulation was observed in a small number of fragmented embryos in CM, sham and control groups. Transferring of these embryos did not lead to any pregnancy. 3.4. Ultrastructure of cytoplasmic fragments The general architecture of embryo fragments was reasonably well preserved (Fig. 2). The main features of cryoinjury, such as plasma membrane disruption, extensive disorganization of the cytoplasm and vacuolization were not observed in these
fragments. The fragments were much smaller than the blastomeres and contained the same complement of normal appearing organelles, when compared with the adjacent blastomeres. In addition, intact and distinct membranes with some remnants of microvilli were observed in almost all of the fragments. Around the fragments, small blebs of cytoplasm and cellular debris were detected. The smooth endoplasmic reticulum (SER) for the most part was vesicular, containing low electron-dense materials. In addition, considerable number of mitochondria were distributed in fragments in comparison to the other cytoplasmic organelles. The majority of mitochondria were intact, rounded to oval in shape and possessed few peripheral cristae with a moderately electron-dense matrix. They were either isolated or associated with SER vesicles forming mitochondria-vesicle (MV) complexes. MV complexes appeared as SER vesicles filled with flocculent and slightly electron-dense material surrounded by mitochondria. The fragments occasionally had remnant of intact undischarged electrondense cortical granules (CGs) beneath the plasma membrane. Annulate lamellae (AL) were occasionally seen and consisted of
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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Table 4 Comparison of clinical outcomes amongst the groups. Variables
Groups CM (n = 30)
Sham (n = 30)
Control (n = 32)
Chemical pregnancy Clinical pregnancy Implantation rate Ongoing pregnancy Delivery rate Live birth rate gender ratio Male Female
8/30 5/30 9/70 5/30 5/30 6/30
8/30 7/30 9/71 7/30 7/30 8/30
12/32 (37.5) 11/32 (34.4) 12/79 (15.2) 11/32 (34.4) 10/32 (31.3) 10/32 (31.3)
0.36 0.19 0.88 0.19 0.40 0.43
5 (50.0) 5 (50.0)
0.41
(26.7) (16.7) (12.9) (16.7) (16.7) (20)
5 (83.3) 1 (16.7)
P value
(26.7) (23.3) (12.7) (23.3) (23.3) (26.7)
5 (62.5) 3 (37.5)
Values are number/total (%).
parallel stacks of cisternae. In addition, remnants of regressing mitochondria, membranes belonging to the Golgi complexes, small clear vacuoles, primary and secondary lysosomes were occasionally found within the fragments. Primary lysosomes were small with dense cores. 4. Discussion The precise etiology of embryo fragmentation is unknown. Various hypotheses have been proposed to explain fragment formation, including an instability within the cortical microfilament network [23], chromosomal abnormalities such as mosaicism and aneuploidy [24] and apoptotic and necrotic processes [25]. In this regard, fragment formation may not have a common origin [23]. In spite of the unknown mechanism underlying the generation of fragments, it is clear that this phenomenon is associated with deleterious ART outcomes [4–8]. Therefore, removal of the embryo fragments have been suggested for enhancing the ART outcomes [19]. Following introducing of embryo fragment removal from 1990s until now, the beneficial effect of fragment removal on low-grade embryos remained as a controversial issue. Only limited retrospective studies have been done in this era that suggested improvement of implantation and clinical pregnancy rates [13,19]. On the other hand, the recent prospective studies did not show any significant improvement in the ART outcomes [18,26,27]. These studies were, however, done on fresh ET cycles and the effect of fragment removal on vitrified-warmed embryos is not known yet. Our prospective randomized study, for the first time, investigated the effects of cosmetic micromanipulation of the cleavage stage embryos on ART outcomes in FET cycles. Also, the potential link between ultrastructural changes of embryo fragments post vitrification was assessed with subsequent effects on ART. The TEM data showed that fragment components including organelles, cytoplasm texture and membrane in vitrified-warmed embryo was well preserved and comparable to the fragments in fresh cleavage embryos [28–30]. In spite of the notion that lysing fragments release toxic materials that can damage the neighboring blastomeres [31], there were no signs of degeneration or lysis due to cryoinjury in our study. Therefore, we conclude that vitrification and warming method did not cause adverse effects on fragments fine structure. In addition, the majority of blastomere’s organelles, except the nucleus, were present in the fragments. This finding supports the opinion that large fragments result in the loss of large volumes of blastomere’s cytoplasm, and deplete the blastomeres from the essential organelles and vital materials, such as mitochondria, mRNA and proteins that can lead to developmental arrest in embryo [32]. Similar to Halvaei’s observation in fresh embryos [26], intact and distinct membrane with some remnants of microvilli were noticed in fragments. In addition, fragments exhibited a uniform cytoplasmic texture with considerable
Fig. 2. Ultrastructure of cytoplasmic fragments in vitrified-warmed embryo by TEM. B, blastomere; M, mitochondria; rM, remnant of regressing mitochondrion; V, SER vesicles; MV, mitochondria-vesicle complex; Ly, primary lysosome; G, membranes belonging to Golgi complexes; mv, microvilli; Va, vacuoles; AL, annulate lamella; CG, cortical granules.
number of mitochondria in comparison to the other cytoplasmic organelles. Our data also indicated that after overnight culturing, the embryos that were subjected to cosmetic micromanipulation, had significantly persistent diminished fragmentation when compared to either sham or control groups. In the CM group, over 90% of the FETs had only 0–5% embryo fragmentations; and in total, none of
Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003
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the ETs in this group involved with embryos >10% fragmentations. The morphological grades improvement was maintained after overnight until ET, which is in agreement with studies on fresh ET cycles [17,18]. The evenness embryos morphology following overnight culturing after cosmetic micromanipulation suggests that the removal of fragments from cleavage cavity and around the blastomeres might modulate the blastomeres division and improve the embryo evenness as hypothesized by Alikani et al. [19]. The fragments positioned in the cleavage cavity may lead to distortion of the division planes or interfere with normal cell–cell contact [19]. In spite of the significant improvements in quality and grade of the embryos after cosmetic micromanipulation and before ET, the clinical outcomes (e.g., implantation, multiple pregnancies, delivery and live birth rates) were not different from the sham and control groups. These findings are in line with the previous prospective investigations on fragment removal in fresh ET cycles [18,27]. In addition, Halvaei and coworkers showed no improvement on the ART clinical outcomes after performing cosmetic micromanipulation on embryos in fresh ET cycles [26]. Besides, coarse granulation was observed in a small number of fragmented embryos in CM and other groups. Transferring these embryos did not lead to any pregnancy in all studied groups, even after granulation removal. The results are also supported by the study of Farhi et al. that showed ET of embryos resulting from oocytes with coarse granulation in their PVS had negative effects on ART outcomes [14]. Lacks of noticeable success in clinical outcomes of transferred embryos post fragment and/or coarse granulation removals in our study and others might be explained by the fact that all of the studies in this era were done on small cohorts [18,26,27], which did not provide strong statistical power to show the exact role of fragment removal in ART outcomes. It also may be linked to several mechanisms that impair or eliminate embryo developmental viability, such as chromosomal abnormalities [33–36], loss of regulatory proteins localized in polarized domains due to fragmentation [23] and loss of essential part of cytoplasm which carries cytoplasmic organelles with fragments [32]. These aforementioned variables are possible reasons for futile effect of fragment removal on clinical outcomes. Also, it was described that embryo development depends on the energy (ATP) production by the mitochondria. Therefore, losing these organelles by fragmentation or exposure of embryo for extended time out of the incubator during fragment removal, may interfere with the viability and implantation potential of the embryos [26]. In conclusion, embryo vitrification had no adverse effects on fine structure of the fragments. In addition, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters, but no noticeable improvements were noticed in ART clinical outcomes. Conflict of interest The authors declare that they have no conflict of interest. Author’s roles SS and IH collected and analyzed the data. SS and SAN interpreted the TEM micrographs. MAK and MA involved in the study design. SS and ZB drafted the article, and all authors critically revised it for important intellectual content. All authors approved the final version of the manuscript. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Acknowledgments This study was extracted from the Ph.D. thesis of Somayyeh Safari. The authors gratefully thank Research and Clinical Center for Infertility, Yazd, Iran, for providing all supports.
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Please cite this article in press as: S. Safari, et al., Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments, Reprod Biol (2017), http://dx.doi.org/10.1016/j.repbio.2017.05.003