Time‐lapse monitoring of mouse embryos produced by injecting sonicated, frozen‐thawed sperm heads with high or low chromosomal integrity

Abstract Purpose To investigate the first‐division kinetics and in vitro development of embryos produced by injecting sonicated sperm heads with high or low chromosomal integrity into oocytes. Methods Mouse spermatozoa were frozen after separating the sperm heads from the tails by sonication in an EGTA solution (EGTA group) or M2 medium (M2 group). The chromosomal integrity of sonicated mouse spermatozoa was analyzed by injecting the sperm heads into fresh mouse oocytes. The developmental potential of spermatozoa was examined by injecting the sperm heads into vitrified‐warming mouse oocytes. We used a time‐lapse monitoring system to compare the first‐division kinetics. Results Chromosomal integrity was preserved significantly more frequently in the EGTA group (90.6%) than in the M2 group (32.7%). Blastocysts developed significantly more often in the EGTA group (80.8%) than in the M2 group (39.6%). In the M2 group, with frequent chromosome aberrations, the time between the sperm injection and first cleavage was delayed (18.4 hours), compared to the EGTA group (16.5 hours). All results of the EGTA group were similar to that of fresh epididymal spermatozoa. Conclusion The EGTA solution for sonication maintained the integrity of sperm chromosomes. Our results revealed a relationship between sperm chromosome integrity and first‐division kinetics.


| INTRODUC TI ON
Intracytoplasmic sperm injection (ICSI) has been widely used, both in reproductive engineering in laboratory animals and for assisted reproductive applications in humans. 1 In 1976, for the first time, Uehara and Yanagimachi reported that injecting human sperm or hamster sperm into hamster oocytes could form a pronucleus. 2 In mice, Kimura and Yanagimachi succeeded in obtaining offspring by performing ICSI with a piezo-micromanipulator. 3 In humans, the first pregnancy achieved with ICSI was reported in 1992. 1 Currently, clinical ICSI applications are used for treating male infertility and IVF failure. In addition, it is possible that ICSI might be used to fertilize oocytes with high probability of success, even with DNAdamaged sperm. Several studies have shown that damage to sperm DNA was associated with fertilization, embryo development, embryo quality, miscarriage after in vitro fertilization (IVF), and ICSI. [4][5][6][7][8][9] Sperm selection is an important problem in modern embryology. However, there is no established method for detecting sperm chromosome damage non-invasively in order to select undamaged sperm for IVF.
With an application of a time-lapse system, embryonic development can be monitored at all times without removing embryos from the incubator. This system enables continuous observation of either embryo morphokinetics or division speed, and thus, these parameters can be measured. 10,11 With a mouse spermatozoon that have a clear sperm chromosome status, it is possible to use the time-lapse system to investigate the effect of sperm chromosome status on embryo development and division speed. This approach could lead to resolution of human male infertility problems.
Sonication of mouse spermatozoa can readily separate sperm tails and heads without using a piezo pulse. 12 However, sonication may increase the frequency of sperm chromosome aberrations. As a matter of course, mouse spermatozoa are highly sensitive to damage. It is thought that the Ca 2+ -dependent DNase is activated by time course after sonication, and activated DNase causes damage to sperm chromosomes. 13 In addition, chromosomal stability in mouse spermatozoa is affected by the sonication medium. It has been reported that suspending mouse spermatozoa in a simple Tris-HCl-buffered solution that contains 50 mmol/L ethylene glycol-bis (-aminoethyl ether) -N, N, N, N -tetraacetic acid (EGTA) and 50 mmol/L NaCl (Tris-HCl EGTA) could maintain chromosome integrity during freeze-drying or freezing without cryoprotection. 14,15 In the golden hamster, sperm chromosome integrity was maintained after sonicating frozen-thawed spermatozoa in Tris-HCl EGTA. 16 However, it is not clear whether this solution might reduce chromosomal abnormalities in mouse sperm heads that are separated from spermatozoa by sonication.
The purpose of present study was to investigate the effect of Tris-HCl buffer with EGTA on the integrity of mouse sperm chromosomes after sonication. Furthermore, we examined the effect of sperm chromosome damage on developmental competence and morphokinetic parameters in mouse oocytes fertilized by ICSI with sonicated frozen-thawed sperm heads.

| Preparation of mouse oocytes
In this study, we used B6D2F1 female mice, 8-12 weeks of age.
At 48 hours after serotropin injection, the female mice received an injection of 5 IU human chorionic gonadotoropin (hCG; Asuka Pharmaceuticals). Cumulus-oocyte complexes (COCs) were collected from oviducts between 15 and 16 hours after the hCG injection.

| Oocyte vitrification and warming
Oocytes were vitrified according to the method described by

| Sperm collection and sperm freezing after sonication
For sperm freezing and sonication, we used M2 medium 18  Spermatozoa were centrifuged at 500 × g for 5 minutes, the supernatant was removed, and spermatozoa were mixed with 1 mL of M2 medium or EGTA solution. This suspension was transferred to ice water (0°C), then sonicated for 5 seconds at 50% sonicator output (20 kHz, VP-5S; Taitec).
More than 90% of mouse spermatozoa underwent head and tail separation. We placed 100-μL aliquots of sonicated spermatozoa, in M2 medium (M2 group) or EGTA solution (EGTA group), into 0.25 mL cryostraws, and froze them in liquid nitrogen (−196°C). Immediately before the piezo-ICSI procedure, the frozen sperm heads were thawed at room temperature (25-28°C).

| Piezo-ICSI procedure
We used an inverted microscope (ECLIPSE Ti-U, Nikon), equipped with a micromanipulator (Narishige Inc.) and a Piezo impact drive Inc.). The inner diameter of the pipette was 5.95 μm, and the outer diameter was 7 μm. Frozen-thawed sperm heads were suspended in 7% polyvinylpyrrolidone (PVP: Irvine Scientific) at a 1:1 ratio before injection. We transferred 10 oocytes into 5 μL droplets of mHTF that contained 10% SSS, which had been placed next to sperm head-containing droplets (10 μL) covered with mineral oil (Irvine Scientific). The zona pellucida was penetrated with a piezo pulse (speed 2, intensity 2); then, to puncture the oocyte plasma membrane, we used one piezo pulse (speed 1, intensity 1). The Piezo-ICSI procedure was completed within 1 hour of sperm thawing. Sperm head-injected oocytes were incubated in 50 μL droplets of global total, under mineral oil, at 37.0°C in 6% CO 2 , 5% O 2 , and 89% N 2 for 96 hours. (v/v) fetal bovine serum) for 7-8 minutes at 37°C. Chromosomes were spread out on clean slides, according to the gradual fixation/ air-drying method. 19 The chromosome slides were stained with 2%

| Preparation and analysis of chromosomes in frozen-thawed sonicated sperm heads
(v/v) Giemsa solution (Merck) in buffered saline solution (pH 6.8) for 8 minutes.

| Time-lapse imaging and annotations
Embryos were individually cultured in an incubator equipped with a time-lapse recording system (PrimoVision, Vitrolife) for approximately 4-5 days, from the ICSI procedure up to the blastocyst stage.

| Statistical analysis
Statistical analyses were performed with GraphPad PRISM 6.03 software (GraphPad Inc.). For binary variables, Fisher's exact probability test and the chi-square test were used to determine statistical differences. For continuous variables, we used the one-way ANOVA and Tukey-Kramer. Significant differences were assumed to be present at P < .05.

| Chromosomal analysis of sonicated, frozenthawed sperm heads
A total of 40 chromosomes per oocyte were judged to be normal ( Figure 1A). The proportion of normal (unaltered) chromosomes was significantly (P < .01) higher in the EGTA group (90.6%) than in the M2 group (32.7%; Table 1). The chromosome normality of the EGTA group was similar to that of fresh epididymal spermatozoa (90.6% vs 97.4%, NS). Thus, the integrity of sperm chromosomes was maintained in the EGTA solution during sonication.
Most chromosome aberrations were structural abnormalities ( Figure 1B). Table 2  and 39.6% vs 80.8%, respectively). In the EGTA group, the proportions of embryos that developed to blastocyst stage were similar to that of fresh epididymal spermatozoa (80.8% vs 88.0% NS).

| D ISCUSS I ON
This study showed that the injection of sperm heads with severe chromosomal damage into oocytes induced delays in the times to reach the PNd and 2-cell stages. We measured the first-division kinetics accurately with time-lapse analysis and revealed a relationship between first-division kinetics and sperm chromosome integrity. In mice, chromosomal abnormalities were previously reported to delay DNA replication, alter the cell cycle, and change the division time. 20,21 The relationship between the recognition and repair of DNA damage in oocytes has been described previously. 22  Note: In this experiment, we used mouse fresh oocytes.
Fresh epididymal spermatozoa use for ICSI before preservation served as controls.  The euploidy of blastocysts should be studied in more detail in future investigations.
In humans, time-lapse analyses showed that the timing of the first division influenced embryonic development. [23][24][25][26][27][28][29][30][31] The mechanism for adjusting the timing of the first division remains incompletely understood. It was shown that the human meiotic spindle characteristics were associated with the first cleavage rate. 32 The relationship between the first-division timing and sperm chromosome integrity in humans remains unclear, but it has been shown that high sperm DNA fragmentation had an effect on the timing of the first division in humans. 33  We found that the EGTA solution could cryopreserve spermatozoa without the addition of a cryoprotection agent. The EGTA solution maintained the integrity of sperm chromosomes, even during sonication. The spermatozoa used for ICSI is typically collected from the minimum number of males possible, and a large number of spermatozoa can be stored in LN 2 without cryopreservation agents.
Sonication does not require a piezo pulse to separate the sperm heads and tails; therefore, ICSI can be completed in a very short time.
In conclusion, we demonstrated that the EGTA solution was useful for maintaining mouse sperm chromosome integrity during sonication. Compared to the EGTA group, the M2 group had a higher rate of chromosomal aberrations after spermatozoa sonication.
Moreover, compared to the EGTA group, the M2 group exhibited significant delays to PNd and to the 2-cell stage after ICSI. As a result, a low percentage of the embryos with severe sperm chromosomal damage developed into blastocysts. These results also indicated that time-lapse monitoring was a useful tool in assessing the PNd and the time to the 2-cell stage for predicting sperm chromosome integrity.

Conflict of interest:
The authors declare no conflict of interest.