Materials and methods. Source of human tripronuclear zygotes. Article - First mitotic spindles in human tripronuclear zygotes - Y-F Gu et al.

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 25 September 2009 Article Analysis of the first mitotic spindles in human in vitro fertilized tripronuclear zygotes after pronuclear removal Yi-Fan Gu began his medical studies in 1997 at Xiangya School of Medicine, Central South University (PR of China) and was awarded his Bachelor s degree in In 2005, he obtained his MSc degree in genetics at the Institute of Reproductive and Stem Cell Engineering, Central South University. During this period, he began his work in human embryology at the IVF laboratory of CITIC-Xiangya Reproductive and Genetic Hospital. From 2006 to date, he has been pursuing his PhD studies, at the same laboratory, in the field of human reproduction and embryology. Mr Yi-Fan Gu Yi-Fan Gu 1, Ge Lin 1,2,3, Chang-Fu Lu 1,2,3, Guang-Xiu Lu 1,2,3,4 1 Institute of Reproductive and Stem cell Engineering, Central South University, Changsha , PR China; 2 CITIC- Xiangya Reproductive and Genetic Hospital, Changsha , PR China; 3 National Engineering and Research Center of Human Stem Cell, Changsha , PR China 4 Correspondence: lugxdirector@yahoo.com.cn Abstract Tripronuclear zygotes (3PN) occur in about 5% of cases in human IVF programmes. Human 3PN zygotes derived from a conventional IVF programme may contain not only the extra male pronucleus but also a supplementary centriole. Researchers have tried to restore diploidy by removing the extra male pronucleus of the tripronuclear zygote. However, it is still unknown whether the procedure can remove the supernumerary centriole. The objective of this study was to evaluate the safety of this manipulation by analysing the first mitotic spindles of 3PN zygotes that have undergone extra pronuclear removal. A controlled trial was conducted using human 3PN zygotes from conventional IVF treatment. In the experimental group, the assumed extra male pronuclei in the 3PN zygotes were removed. The first cleavage patterns and in vitro development were observed in both groups; polarized light microscopy and immunocytochemistry were used to analyse the first mitotic spindles. The blastocyst formation rate was significantly higher (P = 0.007) in the pronuclear-removed group (16.0%) than in the control group (4.5%). No significant differences were found between the groups in the first cleavage patterns and the distributions of the first mitotic spindle structure. This study suggests that, after extra pronuclei are removed, the developmental potential of human 3PN zygotes is improved. However, the abnormal patterns in the first mitosis are not corrected by this removal. Keywords: human, in vitro fertilization, mitotic spindle, polarized light microscope, tripronuclear zygote Introduction Two pronuclei exist in a normal fertilized human zygote, a male and a female. The entry of two or more spermatozoa into an oocyte will cause polyspermic fertilization and the resulting zygotes characteristically display more than two pronuclei after the chromatin of extra spermatozoa decondenses in the ooplasm. Polyspermic fertilization occurs in about 5 7% of cases in human IVF programmes (Pieters et al., 1992; Plachot and Crozet, 1992; Ho et al., 1994; Yie et al., 1996) and most are tripronuclear (3PN) zygotes (dispermic origin). Human tripronuclear zygotes may also result from diploid spermatozoa or digynic origin (oocytes blocked to the meiotic division and diploid oocytes) (Tarín et al., 1999). The extra pronuclei of human 3PN zygotes from conventional IVF have been shown to be of paternal origin in 86% of cases (Kola and Trounson, 1989). Human tripronuclear zygotes (about 3 6%) also occur after intracytoplasmic sperm injection (ICSI) and cytogenetic studies support non-extrusion of the second polar body as the main origin of ICSI-derived 3PN zygotes (digynic origin) (Grossmann et al., 1997). 745 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 746 The majority of human tripronuclear zygotes are triploid (Rosenbusch et al., 1997) and only approximately 6 10% of the 3PN zygotes reach the blastocyst stage (Balakier, 1993; Sathananthan et al., 1999). Although implantation and later development in vivo is possible (Jacobs et al., 1982), these situations typically result in spontaneous abortion; although births of tetraploids and the long survival of triploid children have been reported (Pitt et al., 1981; Sherard et al., 1986), these live births are characterized by severe malformations and multiple anomalies. Therefore, multipronuclear zygotes are excluded from replacement in IVF programmes. Some authors have proposed strategies for correction of triploidy based on the removal of the extra pronucleus according to the position of the second polar body at the zygote stage. Rawlins et al. (1988) first reported the removal of one of the two presumed male pronuclei from human 3PN zygotes to restore the diploid state, but syngamy occurred in one of three enucleated 3PN zygotes and cleavage was not observed. Gordon et al. (1989) achieved a 55% survival rate of 3PN zygotes after pronuclear removal with the same technique. At an early stage, cytochalasin B (cytoskeletal relaxing agent) was usually used to assist pronuclear removal. Malter and Cohen (1989) reported pronucleus removal in the absence of cytoskeletal relaxants and achieved a 36% survival rate. Palermo et al. (1994) reported pronucleus removal by puncturing the zygote s plasma membrane with a sharp needle and inserting it into the target pronucleus and provided evidence that the human sperm centrioles control the first mitotic divisions after fertilization. Iviakheneko et al. (2000) showed high efficiency in removing the extra pronuclei from human 3PN zygotes with blunt pipettes in the absence of cytochalasin B and 100% of the 3PN zygotes survived. Feng and Gordon (1996) reported the birth of normal mice after the removal of supernumerary male pronuclei from polyspermic zygotes treated with cytochalasin. Kattera and Chen (2003) reported that after the microsurgical enucleation of a single pronucleus from each of three tripronuclear zygotes was performed and the embryos were transferred to a 38-year-old woman on day 3 after fertilization, a normal healthy baby boy was born. Escribá et al. (2006) reported that heteroparental blastocysts were derived from human tripronuclear zygotes after extra pronuclear removal. The mitotic spindle apparatus is composed of a pair of centrioles at the poles, the microtubule and the chromosome plates. It is important to maintain the normal genome ploidy in a human embryo during cleavage and further development. The centriole is the main microtubule-organizing centre (MTOC) in mammals (Schatten, 1994). In mice, the spermatozoon does not bring a functional centriole into the zygote (Schatten et al., 1986), so murine dispermic zygotes only contain the extra male pronuclei, and it is theoretically feasible to correct this condition by pronuclear removal. In humans, the centriole of the zygote is paternally inherited from the fertilizing spermatozoon (Sathananthan et al., 1991). Human dispermic zygotes derived from a conventional IVF programme contain not only the extra male pronucleus but also a supplementary centriole. This presence frequently results in a tripolar spindle or, less often, in the formation of a bipolar spindle when the supernumerary centrosome remains dormant (Plachot et al., 1989; Staessen and Van Steirteghem, 1997; Sathananthan et al., 1999). The segregation of the three sets of chromosomes on a bipolar spindle results in a uniformly triploid 2-cell embryo. However, tripolar spindle formation results in a mosaic embryo with three equally sized blastomeres at the first mitotic division, due to the random segregation of the sister chromatids of the three haploid sets of chromosomes to the three poles (Palermo et al., 1994; Staessen and Van Steirteghem, 1997; Sathananthan et al., 1999). Tripolar spindles at cleavage and the early blastocyst stages could also result in chromosomal malsegregation and explain the chromosomal chaos identified by multicolour fluorescent in-situ hybridization (FISH) analysis in some cleavage stage embryos (Delhanty et al., 1997). Whether the pronuclear-removal procedure can remove the supplementary centriole and restore the normal mitotic spindle is still unknown. There has been no direct evidence so far to prove that this procedure is able to correct the number of centrioles in 3PN zygotes and maintain the normal mitotic spindle at the first cleavage. Most studies image the mitotic spindle using immunofluorescence or electron microscopy, which require samples to be fixed in advance. These methods illuminate the detailed structure of the mitotic spindle, but they have limited value in analysing the dynamics of mitotic spindle and samples also lose further development potential after fixation. A polarized light microscope was developed to study the birefringence of living cells (Oldenbourg and Mei, 1995; Oldenbourg, 1996) and has been applied to mammalian embryology (Wang and Keefe, 2002). The polarized light microscope uses novel electro-optical hardware and digital processing to image macromolecular structures in cells on the basis of their birefringence (an inherent physical property of highly ordered molecules such as microtubules). It provides a new opportunity to non-invasively investigate spindle dynamics. In previous studies, polarized light microscopy has been employed for the non-invasive visualization of meiotic spindles in human oocytes (Wang et al., 2001). Until now, few studies have investigated mitotic spindles in early human embryos using a polarized light microscope. The objective of this study was to evaluate the safety of the extra pronuclear-removal manipulation reported in previous studies by analysing the first mitotic spindles and evaluating the in vitro developmental potential of human 3PN zygotes after extra pronuclear removal. Materials and methods Source of human tripronuclear zygotes In this study, 3PN zygotes were donated from patients who received conventional IVF treatment at Reproductive and Genetic Hospital of CITIC-Xiangya. Signed informed consent was obtained from these patients before performing

3 experiments. This study was approved by the ethical committee at the local hospital. In-vitro fertilization and pronuclear assessment All patients underwent ovarian stimulation induced by rfsh (Gonal-F; Serono, USA), after pituitary down-regulation by the administration of GnRH agonist (Leuprorelin; Takeda, Japan). GIII series sequential culture system (Vitrolife, Sweden) was used for in vitro culture of human oocytes and embryos, according to the manufacturer s instructions. On day 0, oocyte retrieval was performed by ultrasound-guided transvaginal aspiration 36 h after human chorionic gonadotrophin (Profasi; Serono) administration ( ,000 IU). Cumulus oocyte complexes (COC) were collected in 1 ml G-FERT medium (Vitrolife) and cultured for 3 6 h to induce full maturation of oocytes. In the conventional IVF programme, COC were inseminated with prepared spermatozoa (final density about 10 5 / ml) and cultured in fresh G-FERT medium (2 3 COC/ 1 ml) to the next day. After h post-insemination on day 1, all oocytes were denuded with hand-pulled pipettes (about 150 lm inner diameter) and examined for the number, shape and distribution of pronuclei and nucleoli using the pronuclear scoring system, modified from Scott and Smith (1998) under an inverted microscope (TE-2000U; Nikon, Japan) equipped with Hoffman modulation optics. The 3PN zygotes (with three distinct pronuclei, the first and the second polar body) were selected out and cultured in 10 ll/drop G1.3 medium (Vitrolife) separately for further experiment. All 3PN zygotes were allocated randomly to the pronuclearremoved group or the control group according to the patient record number. Pronuclear removal and embryo culture Two types of handmade microtools were used for micromanipulation: a holding pipette (outer diameter, 100 lm; inner diameter, 20 lm) and a blunt enucleation micropipette (inner diameter: 10 lm). To accommodate the micro-manipulation set-up, both microtools were bent to a 35 angle. All manipulations were performed at 37 C in G-MOPS (Vitrolife) handling medium (5 ll/drop) on an inverted microscope equipped with two hydraulic micromanipulators (Narishige, Japan). The Tefion tubing for the hydraulic systems was filled with light mineral oil. Pronuclear removal (Figure 1) was performed within 1 2 h after pronuclear assessment on day 1, according to a modified protocol already described (Iviakheneko et al., 2000). Briefly, the 3PN zygote was gently rotated with a holding pipette until the assumed male pronucleus was at a 3 o clock position, which is furthest from the second polar body, and a hole was made on the zona close to the targeted pronucleus by a laser drilling system (ZILOS-tk; Hamilton Thorne, USA). An enucleation micropipette was inserted through the hole into the perivitelline space and pressed on the plasma membrane. The assumed male pronucleus was aspirated entirely into the enucleation micropipette and gently pulled until the cytoplasmic bridge pinched off. The integrity of the removed pronucleus, the remaining two pronuclei and the cytoplasmic membrane was examined to confirm whether the pronuclear removal was successful. Those 3PN zygotes with unsuccessful pronuclear removal including zygote degeneration, incomplete pronuclear removal or more than one pronuclei removed were excluded from the experiment. These 3PN zygotes with successful pronuclear removal were cultured in G1.3. In the control group, intact 3PN zygotes were cultured in the same conditions. All embryos were transferred to fresh G1.3 and cultured until day 3. They were then transferred into fresh G2.3 medium (Vitrolife) to support the blastocyst growth on the afternoon of day 3. Their development was checked every day and the blastocyst formation rate was followed until day 5. Embryos were evaluated according to the conventional criteria with high-grade embryos defined by: (i) number of blastomeres: at least six on day 3; (ii) shape of blastomeres: almost equal; (iii) fragmentation 20%; and (iv) blastomeres transparent without serious cytoplasmic inclusion or vacuoles. Culture media were supplemented with 5% human serum albumin (Vitrolife) and covered under paraffin oil (Vitro- Figure 1. Pronuclear removal of human 3PN zygotes. (A) Human 3PN zygote. (B) Human 3PN zygote after pronuclear removal. 400 magnification. 747

4 748 life) and pre-equilibrated overnight. All embryos were cultured at 37.5 C/6% CO 2 /5% O 2 with 100% humidity. Each embryo in both groups was cultured in a medium drop (10 ll) individually to distinguish them. Polscope analysis of the first mitotic spindle To minimize the potential influence of in vitro observation, a subset of samples were examined for the first mitosis. After 20 h post-insemination on day 1, 3PN zygotes in both groups were observed every hour until their pronuclei disappeared (syngamy). When their pronuclei disappeared, the first mitosis began. During the period, the first cleavage patterns were recorded and the first mitotic spindles were observed by polarized light microscopy (Polscope). To image the first mitotic spindle, each sample was placed individually in G- MOPS handling medium (10 ll) covered with warm paraffin oil in a glass-bottomed (0.17 mm thick) Petri dish (20 mm diameter) (Delta TPG dish; Bioptechs, USA).Samples were examined with an inverted microscope with Hoffman interference optics 20 objective lenses equipped with LC Polscope filters, analogue video camera (COHU; Cambridge Research and Instrumentation, USA) and a personal computer (Dell, USA) running image analysis software (LC-Polscope pro 4.4; Cambridge Research and Instrumentation). Dishes were maintained at 37 C during examination with a thermal plate (IVF-1200; Pacific Contrast Co, USA) and an objective heater (HT 300; Minitüb, Germany). Alignment of the microscope and calibration of the software were performed before imaging. The first mitotic spindles were imaged at least five times from different directions by rotating samples at an equatorial plane of each embryo to ensure the accuracy of three-dimensional information of mitotic spindles. Digital images were saved to a compact disk and the best images were selected for subsequent analysis. Microtubule orientation in the first mitotic spindle was analysed and confirmed by image analysis software of Polscope (function Display Retardance/Azimuth Composite Image ). All warming plates were calibrated previously to ensure that samples were strictly kept at 37 C during observation and all results were collected by the same observer. The duration of mitotic spindle observation was min in each sample. After observation, samples were cultured further to observe the first cleavage in the above conditions or fixed for immunocytochemistry according to experimental design. Immunocytochemistry analysis of the first mitotic spindle The first mitotic spindle morphology and chromosomal alignment in both groups were also evaluated by indirect immunocytochemistry, according to a modified protocol already described (Messinger and Albertini, 1991). All reagents and antibodies were purchased from Sigma. Zona pellucida was removed by a brief incubation (0.5 1 min) at 37 C with acidic Tyrode s solution (ph = 2.5) and zona-free zygotes were fixed and permeabilized for 30 min at 37 C in a microtubule stabilizing buffer (0.1 mol/l PIPES, ph = 6.9, 5 mmol/l MgCl 2.6H 2 O, 2.5 mmol/l ethylene glycol-bis EGTA containing 2.0% formaldehyde, 0.5% Triton X-100 and 1 lmol/l taxol). Fixed zygotes were blocked for overnight at 4 C with 2% bovine serum albumin, 2% normal goat serum, 0.1 mol/l glycine and 0.01% Triton X-100 in phosphate-buffered saline (PBS). To analyse the mitotic spindle, samples were incubated with 1/200 anti-a-tubulin monoclonal antibody in PBS + bovine serum albumin overnight at 4 C. After primary antibody incubation, the samples were washed three times in 0.05% Tween in PBS and incubated at 37 C in blocking solution for 1 h. Samples were incubated in 1/100 goat anti-mouse tetramethylrhodamine B isothiocyanate-conjugated secondary antibody for 1 h at 37 C. After three washes in 0.05% Tween in PBS, samples were counterstained with DAPI for 5 min at room temperature, washed in PBS and mounted between a slide and a coverslip in mounting medium (glycerol in PBS; Sigma). Samples were examined by conventional fluorescence microscope (TE-2000U) and laser scanning confocal microscope (FV-1000, Olympus, Japan). The images were processed by Adobe Photoshop version 7.1 software (Adobe Systems, USA). The results from Polscope and indirect immunofuorescent staining in the same samples were used to evaluate the accuracy of Polscope analysis. Statistical analysis The first cleavage pattern, in vitro development potential and the first mitotic spindles between the pronuclearremoved and the control group were analysed using the chi-squared test. The accuracy of Polscope analysis was analysed using the paired chi-squared test and Kappa test. Significance was considered when P < Results In the first stage of the investigation, in vitro development potential was observed in the pronuclear-removed 3PN and intact 3PN groups. In the second stage, first mitosis was investigated in additional 3PN zygotes. A summary of the numbers of 3PN zygotes used in the different stages of the investigation is presented in Table 1. In vitro developmental potential of extra pronuclear-removed and intact human 3PN zygotes A total of 211 3PN zygotes were allocated in both groups for in vitro development observation. Fifteen 3PN zygotes with unsuccessful pronuclear removal were excluded in this study, including zygote degeneration (n = 12), incomplete pronuclear removal (n = 2) or more than one pronucleus removed (n = 1) and 196 3PN zygotes in both groups were observed for in vitro development (Table 2). Of 98 pronuclear-removed human 3PN zygotes, 83 (84.7%) were manipulated successfully, 81 (97.6% of those with successful PN removal) cleaved, 59 (72.8%) became high-grade embryos on day 3, and 13 (16.0%) reached the blastocyst stage on day 5. Of 113 intact human 3PN zygotes, 110 (97.3%) cleaved, 89 (80.9%) became high-grade embryos (cell number 6, fragments 20%) on day 3, and five (4.5%) reached the blastocyst stage on day 5. No significant difference was

5 Table 1. Number of tripronuclear (3PN) zygotes used in the study. Pronuclear-removed 3PN Intact 3PN Stage one In vitro development (conventional inverted microscope) Stage two Mitotic spindle analysis (Polscope) Fixation for confocal (two samples lost) First cleavage observation Mitotic spindle analysis (confocal) Fixation after Polscope analysis Direct fixation (Polscope occupied) 2 11 Table 2. In vitro development of human 3PN zygotes with or without pronuclear removal. Pronuclear-removed 3PN Intact 3PN P-value Number Successful PN removal a 83 (84.7) Cleavage b 81 (97.6) 110 (97.3) NS High-grade c 59 (72.8) 89 (80.9) NS Blastocyst d 13 (16.0) 5 (4.5) Values are number (percentage). NS = not significant. PN = pronucleus. Chi-squared test was used to determine significant differences between groups. a Expressed as the number (percentage) of survived 3PN zygotes to the number of 3PN zygotes before manipulation. b Expressed as the number (percentage) of cleavage embryos to the number with successful PN removal or intact PN zygotes. c Expressed as the number (percentage) of high-grade embryos relative to the number of cleavage embryos. d Expressed as the number (percentage) of blastocysts relative to the number of cleavage embryos; P = found in cleavage rate and the percentage of high-grade embryos between the groups. The blastocyst formation rate was significantly higher (P = 0.007) in the pronuclearremoved group than in the control group. The first cleavage pattern in both groups A total of 44 human 3PN zygotes in both groups were examined for their first cleavage patterns (Table 3). In the first cleavage, two patterns existed in both groups: (i) cleavage into two daughter cells and (ii) cleavage into three daughter cells at the first mitosis. There was no significant difference between the pronuclear-removed group (cleavage to two cells = 15, cleavage to three cells = 7) and the intact 3PN group (cleavage to two cells = 13, cleavage to three cells = 9). Polscope analysis of the first mitotic spindles The first mitotic spindles were examined using a Polscope in a total of 90 human 3PN zygotes in both groups (Table 3). During the first mitosis, the Polscope results showed that various abnormal spindles existed in both groups (Figure 2). In the pronuclear-removed group (n = 42), 18 (42.9%) showed a single bipolar spindle structure (uniform microtubule orientation) and the other 24 samples (57.1%) showed abnormal spindle conformation (multipolar microtubule orientation or multi-spindle). In the control group (n = 48), 15 (31.3%) showed a single bipolar spindle structure and the other 33 samples (68.8%) contained similar abnormalities. There was no significant difference between the groups. In the first cleavage, the human 3PN zygotes with bipolar mitotic spindles were seen to cleave to 2-cell embryos and human 3PN zygotes with typical tripolar mitotic spindles cleaved to 3-cell embryos in both groups. Immunocytochemistry analysis of the first mitotic spindles The first mitotic spindles in a total of 57 human 3PN zygotes in both groups were examined using an indirect immunofluorescent method (Table 3). All samples analysed by immunofluorescent staining were not included in the calculation of in vitro development observation due to further loss of development potential after fixation. At the end of the pronuclear stage, microtubules began to assemble around the pronuclei in intact 3PN zygotes and two sperm tails were observed around the two male pronuclei, which were far from the second polar body (Figure 3). During the first mitosis, the immunocytochemistry results revealed 749

6 Table 3. First cleavage and mitotic spindle of human 3PN zygotes with or without pronuclear removal. Pronuclear-removed 3PN Intact 3PN First cleavage patterns n =22 n =22 Cleavage to two cells 15 (68.2) 13 (59.1) Cleavage to three cells 7 (31.8) 9 (40.9) First mitotic spindle (Polscope analysis) n =42 n =48 Bipolar spindle 18 (42.9) 15 (31.3) Abnormal spindle 24 (57.1) 33 (68.8) First mitotic spindle (immunocytochemistry analysis) n =22 n =35 Bipolar spindle 9 (40.9) 13 (37.1) Abnormal spindle 13 (59.1) 22 (62.9) Values are number (percentage). PN = pronucleus. There were no statistically significant differences between the two groups (Chi-squared test). Figure 2. Polscope analysis of the first mitotic spindles in (A and C) human 3PN zygotes and (B and D) 3PN zygotes with pronuclear removal. (A and B) Normal bipolar spindle; (C and D) tripolar spindle. 200 magnification. 750 various abnormal spindles and chromosomal alignments in both groups (Figure 4). In the pronuclear-removed group (n = 22), nine (40.9%) showed a single bipolar spindle structure and regular chromosome alignment and the other 13 samples showed tripolar spindle (n = 9), multi-spindle (n = 3) or irregular spindle (n = 1); whereas, in the control group (n = 35), 13 (37.1%) had normal spindle structure and chromosome alignment and the other 22 samples showed similar abnormalities, including tripolar spindle (n = 17), multi-spindle (n = 3) or irregular spindle (n = 2). There was no significant difference between the groups. Accuracy of Polscope analysis In 44 samples from both groups, the first mitotic spindles were analysed using a Polscope and an indirect immunofluorescent method, successively. The results were compared and 37 (84.1%) were consistent between the methods (Kappa value = 0.67), indicating moderate agreement. Discussion Most human tripronuclear preimplantation embryos show a normal development in vitro until the first two or three cell cycles. Thereafter, they exhibit retarded, arrested or abnormal development, which is revealed by unequally sized blastomeres, cellular fragmentation, multinucleation and spindle defects (Sathananthan et al., 1999). Only approximately 6 10% of the 3PN zygotes reach the blastocyst stage (Balakier 1993; Sathananthan et al., 1999). In the present study, similar results were found. At the cleavage stage (from day 1 to day 3), 3PN zygotes showed almost-normal morphological features: high cell number

7 directly to three cells at the first cleavage division. These embryos have a severely abnormal (but not triploid) chromosomal complement. Furthermore, some (4/29, 14%) tripronuclear human oocytes cleave to two cells plus an extrusion and these embryos are diploids; whereas, some (7/29, 24%) cleave to two cells and these embryos are triploid after the first cleavage division. Figure 3. Confocal laser scanning microscopy images of the microtubules polymerizing in human 3PN zygotes. The microtubules begin to polymerize around pronuclei in human 3PN zygotes to form a tripolar spindle. Two sperm tails are attached to two male pronuclei, which are far from the second polar body. Red = a-tubulin; blue = DNA. 600 magnification; scale bar = 40 lm. and cleavage rate, equal blastomeres, low cytoplasmic fragments and a high embryo grade. Most 3PN zygotes arrested on day 4 or day 5 and only 4.5% reached the blastocyst stage. In humans, embryonic genome activation occurs at the 4- to 8-cell stage (Braude et al., 1988). Cell cycle checkpoints may not operate at these early stages (Delhanty and Handyside, 1995), which could explain the almost-normal development of human 3PN zygotes at the cleavage stage. Genes regulating chromosome segregation, the cell cycle and apoptosis express maximally at the blastocyst stage of human preimplantation development (Wells et al., 2005). The mechanisms of the cell cycle checkpoints and programmed cell death may detect abnormalities in human embryos and prevent them from progressing to the blastocyst stage (Musacchio and Hardwick, 2002; Jurisicova and Acton, 2004). Researchers have observed and analysed the first cleavage pattern of human tripronuclear zygotes at an early stage. Angell et al. (1986) conducted the first direct cytological observations of the behaviour of tripronuclear zygotes. The chromosome studies and DNA measurements of 3PN embryos showed that about one-half were triploid; the others had either a normal diploid chromosome complement or a severely depleted chromosome complement. Kola et al. (1987) analysed the cleavage pattern during the first mitotic division of human 3PN zygotes and the chromosomal composition of the resulting preimplantation embryos. This study provided evidence, for the first time, that most (18/29, 62%) tripronuclear human oocytes cleave Three main developmental outcomes of human 3PN zygotes were later confirmed and expanded upon by other studies (Plachot et al., 1987, 1992; Pieters et al., 1992; Zenzes and Casper, 1992; Ma et al., 1995; Rosenbusch et al., 1997; Tarín et al., 1999). Golubovsky (2003) summarized the variants of human 3PN zygote cleavage and their possible developmental outcomes as follows: (i) a mitotic division with bipolar spindle, giving 3PN blastomeres and embryos in about 25% of cases; (ii) exclusion of one haploid genome from the metaphase plate of the first cleavage division (14 32% of cases), resulting in 2PN diploid, 2PN/3PN mosaics and 1PN/2PN derivatives; and (iii) in 50 60% of 3PN zygotes, a tripolar spindle is formed at the first cleavage division, resulting in dramatic abnormalities in chromosome distribution. However, these conclusions were reached from previous cytogenetic results and these researchers did not directly observe the mitotic spindles of human 3PN zygotes at the first cleavage. In the present study, a polarized light microscope was used to continually observe the first cleavage of human 3PN zygotes. The authors also observed that two patterns (cleavage to two cells and cleavage to three cells) occurred in the first cleavage of human 3PN zygotes. These results were consistent with previous studies. In addition, human 3PN zygotes with bipolar mitotic spindles were directly observed (using Polscope) to cleave to 2-cell embryos; 3PN zygotes with tripolar spindles cleaved to 3-cell embryos. These observations proved that abnormal mitotic spindles resulted in abnormal cleavage patterns. In this study, two cells plus an extrusion was not considered to be a special cleavage pattern, as an extrusion could not be distinguished from a smaller blastomere, a cytoplasmic fragment or a larger polar body based on morphological differences without cytogenetic analysis. To further clarify the first mitotic spindle abnormalities in human tripronuclear zygotes, polarized light microscopy and immunocytochemistry staining were performed. Various abnormal spindles were detected in human 3PN zygotes in the first cleavage, including tripolar spindles, multi-spindle and irregular-shape spindles. These results indicate that numerous MTOC abnormalities exist in human 3PN zygotes. Human 3PN zygotes have dispermic or digynic origin after conventional in vitro fertilization. The authors postulate that one or two centrioles are introduced into human 3PN zygotes and that their different behaviours result in the various patterns of the first mitotic spindles: (i) if two centrioles are introduced and one duplicates and the other does not, it will result in three active MTOC and a tripolar mitotic spindle; (ii) if both introduced centrioles duplicate and separate, it will result in four active MTOC and two spindles (multi-spindle); and (iii) if both centrioles do not duplicate or if only one centriole is 751

8 Figure 4. Confocal laser scanning microscopy images of the first mitotic spindles in (A, C, and E) human 3PN zygotes and (B, D, and F) 3PN zygotes with pronuclear removal. (A and B) Normal bipolar spindle; (C and D) tripolar spindle; (E and F) multi-spindle. Red = a-tubulin; blue = DNA. 600 magnification; scale bar = 40 lm. 752 introduced and duplicates, it will only produce two active MTOC and a bipolar spindle. Many previous researchers expected to correct human tripronuclear zygotes by extra pronuclear removal and, thereby, obtain normal human embryos. Recently, Escribá et al. (2006) reported that after extra pronuclear removal, 41% (21/51) of the manipulated embryos developed in vitro to the blastocyst stage, which was significantly higher than in the control group (8/42, 19%). FISH analysis performed on eight manipulated embryos confirmed their diploid state (four blastocysts were diploid, two were mixploid with similar percentages of coexisting diploid and tetraploid cell lines, one had a tetraploid complement and one had no informative FISH result); three out of four controls were triploid (one had no informative FISH result). Heteroparental inheritances were also confirmed in four of six manipulated embryos. In the present study, the authors analysed the in vitro development potential and the first mitotic spindles in human 3PN zygotes with pronuclear removal, in order to evaluate

9 the risk of the procedure. After removing the extra pronucleus, the blastocyst formation rate was significantly elevated and 16% of the 3PN zygotes reached the blastocyst stage. It should be noted that this result was lower than that of normal fertilized embryos (40%, unpublished data) in this group s laboratory. The results indicate that the pronuclear-removal procedure improves the development potential of 3PN zygotes in a limited manner. After the extra pronucleus is removed, the part of the 3PN zygote that formed the bipolar mitotic spindle during the first cleavage may produce diploid (2n) embryos. Abnormalities of these 3PN zygotes may be improved and they have more development potential to reach the blastocyst stage. Additionally, Golubovsky (2003) supposed that post zygotic diploidization of triploids could better explain cytogenetic events in 3PN embryos. According to this theory, the extra pronuclear removal may increase the percentage of 2n cells in 2n/3n mixploid embryos and diploidization of these embryos may increase their potential to reach blastocyst stage. In the experimental group, two patterns (cleavage to two cells and cleavage to three cells) were found to occur at the first cleavage and no significant difference was found between the groups. The first mitotic spindles were further investigated in the experimental group. There was no significant improvement of the first mitotic spindles and similar spindle abnormalities existed after pronuclear removal. These results indicate that the pronuclear-removal procedure may not eliminate the supernumerary centriole and restore the normal structure of the mitotic spindle apparatus. In conclusion, the present study showed that pronuclear removal cannot correct mitotic spindle abnormalities in human tripronuclear zygotes from a conventional IVF programme. The safety of this manipulation is not assured. In addition, male and female pronuclei in human zygotes may not be discriminated by size or, unambiguously, by position. Although the female pronucleus tends to be closer to the second polar body, the presence of a third pronucleus is likely to perturb the normal cell organization at that stage, making even more arduous the task of identifying male and female pronucleus. Manipulators could not exclude the chance that their micromanipulation methodology may lead to the removal of the female (rather than one of the male) pronucleus. In addition, it is also not easy to distinguish the dispermic and digynic 3PN zygotes by morphology, because the first polar body is similar to the second polar body in some samples. Therefore, the accuracy of correctly removing the extra pronuclei in human 3PN zygotes is also not assured. Considering these reasons, the authors do not propose to transfer these corrected 3PN zygotes to patients, in spite of a successful case report (Kattera and Chen, 2003). Although the authors are wary about using these embryos in a clinical setting, the corrected 3PN zygotes could be a possible source of human embryonic stem cell derivation. Centrosomal defects can cause embryonic arrest through the formation of abnormal spindles and the accumulation of chromosomally abnormal cells that derive from them (Chatzimeletiou, 2008). These abnormalities occur not only in abnormal and arrested embryos but also in normal developing embryos (Chatzimeletiou et al., 2005). Mitotic spindle abnormalities may be a major pathway leading to post zygotic chromosomal abnormalities, which may account for the developmental loss in human embryos (Los et al., 2004). The polarized light microscopy provides a rapid, simplified and non-invasive method for identifying mitotic spindles in human embryos. 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