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. 2009 Apr 8;8(2):63–69. doi: 10.1007/s12522-009-0010-4

Embryo development after intracytoplasmic sperm injection can be predicted by assessment of sperm nuclear chromatin

Tomoko Takayama 1,✉, Haruo Katayose 2,✉, Kaoru Yanagida 2, Akira Sato 1
PMCID: PMC5904575  PMID: 29699309

Abstract

Purpose

To assess the influence of structural differences in sperm nuclei on embryo development in intracytoplasmic sperm injection (ICSI).

Methods

Semen obtained from forty‐four infertile patients who underwent ICSI was examined. In assessing blastocyst development, only those patients who had successfully obtained over five fertilized eggs were included to exclude any oocyte factors (n = 22). Spermatozoa were assessed using excitation fluorescence flow cytometry with acridine orange and the sperm chromatin dispersion (SCD) test.

Results

There was a significant positive correlation between the COMP values obtained from flow cytometry and blastocyst formation. (r = 0.477, p = 0.025). There was a significant negative correlation between the SCD values representing DNA fragmentation and blastocyst formation. (r = 0.796, p < 0.001). COMP values and SCD values were independent parameters to assess sperm nuclear quality regarding embryo development in vitro (r = 0.224, p = 0.080).

Conclusion

Results suggest that injection of spermatozoa with fewer disulfide bonds and less nuclear DNA fragmentation could achieve better blastocyst formation in human ICSI. Assessment of sperm chromatin should help to predict embryo development after ICSI.

Keywords: Sperm chromatin, Disulfide bonds, DNA fragmentation, ICSI, Embryo development

Introduction

Deterioration of sperm profiles, such as decreased concentration or motility, has been reported recently [1];, at the same time, demand for intracytoplasmic sperm injection (ICSI) has been increasing [2]. Eighteen to 40% of infertility is caused by male factors [3], however, 30% of them are idiopathic in spite of their normal semen profiles [4]. Studies using acridine orange (AO) fluorescent dye have revealed that human fertility is influenced by structural nuclear proteins of the sperm head [5]. Initial studies have supported a role for chromatin alterations in unexplained infertility [6].

With regard to the influence of spermatozoa on embryo development in vitro, although some reports to the contrary can be found in the literature, rate of blastocyst formation after ICSI has been recognized as lower than that after conventional in vitro fertilization (c‐IVF) [7, 8, 9, 10, 11, 12]. It can be assumed that by‐passed acrosome reaction and sperm–egg fusion along with simultaneous incorporation of the sperm cytoplasmic membrane have adverse effects on the synchronized process of fertilization needed for subsequent adequate embryo development [13, 14]. Furthermore, spermatozoa having aberrant nuclear chromatin, such as immature structure or damaged DNA, can be detected among spermatozoa with extensive nuclear heterogeneity under 200×–300× magnification.

The aim of this study was to assess by clinical study the influence of structural differences in sperm chromatin on embryonic development after ICSI.

Materials and methods

Quality assessments of sperm nuclei

Forty‐four infertile couples who underwent ICSI at our hospital between January 2004 and November 2006 agreed to participate in this study. This study was approved by the Fukushima Medical University Institutional Review Board. Ejaculated sperm were collected by masturbation from 44 patients. Conventional semen parameters were assessed by computer‐assisted sperm analysis (CEROS V12, Hamilton Thorne Research, MA, USA). Severe male infertile factor, such as cryptozoospermia or azoospermia, were excluded from this study. Liquefied semen was mixed with HEPES‐buffered human tubal fluid (HTF) (Irvine, USA) and centrifuged at 300×g for 5 min to remove seminal plasma. Resultant pellets were resuspended in 1 ml of medium at a final concentration between 50 × 106 and 100 × 106/ml. Sperm suspensions were divided into two groups, one for chromatin assay and the other for DNA fragmentation study. For chromatin assay, 0.2 ml of the suspension was mixed with an equal volume of 10 mM N‐ethylmaleimide (NEM, SIGMA, USA) in PBS to avoid natural oxidation of nuclear proteins (protamine). An additional 0.2 ml of the suspension was stored for DNA fragmentation study. All aliquots were stored at −80°C until measurement.

Sperm chromatin status was assessed according to the method of Spano et al. [15] with minor modifications. After thawing at room temperature, aliquots were dissolved to 0.2 ml of sperm suspension in PBS. Each aliquot was mixed with 0.4 ml low pH detergent solution (0.17% TritonX‐100 (SIGMA), 0.15 M NaCl (WAKO, Japan) and 0.08 N HCl (WAKO), pH 1.4) for 30 s followed by staining with 1.2 ml of AO solution (6 mg/l AO (3,6‐bis[Dimethylamino]acridine, hemi[zinc chrolide]salt, A‐6014, SIGMA), 0.1 M citrate (SIGMA), 0.2 M Na2HPO4 (WAKO), 1 mM EDTA (ethylenediaminetetraacetic acid, disodium salt, STRATAGENE, CA, USA), and 0.15 M NaCl (pH 6.0)) for 3 min. After filtering through a nylon mesh (50 μm), spermatozoa in each aliquot were assessed by flow cytometry using a FACScan cytometer (soft; CELL Quest, Becton Dickinson Immunocytometry Systems, CA, USA). 10,000 spermatozoa in each sample were analyzed with excitation at 488 nm. Cells within a gate defined by forward scattered light and side scattered light were counted (Fig. 1a). Prior to assessment, gated cells were confirmed to be sperm nuclei by using dithiothreitol (DTT, 5 mM in PBS; SIGMA), a reducing agent, or diamide (5 mM in PBS; SIGMA), an oxidant of thiol groups (SH) which could alter the emission pattern from sperm nuclei. Cytograms (as in [15]) were obtained by plotting the fluorescence intensity of each cell. The cells outside of the main population (COMP, %) were utilized as an index of this study [6, 15] (Fig. 1b, c).

Figure 1.

Figure 1

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a Gate (circle) enclosing spermatozoa, which was defined by forward scattered light and side scattered light in flowcytometry. The vertical axis is intensity of side scattered light and the horizontal axis is that of forward scattered light. b, c Cytograms obtained from same sample. The vertical axis (FL‐1) is intensity of green fluorescence and the horizontal axis (FL‐3) is that of red fluorexcence. Fluorescent intensity changed from a to b by DTT. COMP (%) was defined as a percentage of the number of spermatozoa within the line (area R)

To assess DNA fragmentation, the sperm chromatin dispersion (SCD) test reported by Fernandez et al. [16] was performed with some modifications. After thawing, 0.2 ml of the sperm suspension was diluted in PBS to the concentrations between 25 × 106 and 50 × 106/ml. 100 μl of the suspension was mixed with an equal volume of 1.4% low‐melting agarose (NUSIEVE GTG, FMC BioProducts, ME, USA). Each mixture (0.05 ml) was pipetted onto glass slides precoated with 0.65% standard agarose (SeaKem GTG, Cambrex Bioscience Rockland, ME, USA), then covered with coverslips (18 × 18 mm). After allowing these aliquots to solidify at 4°C for more than 4 min, coverslips were removed carefully, and slides were immediately immersed in an acid solution (0.08 N HCl) for 7 min at room temperature. Nuclear proteins were removed by transferring the slides into lysing solution A (0.4 M Tris (TRIZMA BASE, SIGMA), 0.8 M DTT, 1% sodium dodecyl sulfate (SDS, Bio‐Rad Laboratories, CA, USA), and 50 mM EDTA (pH 7.5)) for 10 min, with a subsequent immersion in lysing solution B (0.4 M Tris, 2 M NaCl, and 1% SDS (pH 7.5)) for 5 min at room temperature. Slides were washed three times in Tris–EDTA buffer (0.4 M Tris and 2 mM EDTA (pH 7.5)) for 2 min, then dehydrated by sequential 2 min exposure in 70, 90, and 100% ethanol (WAKO). After drying in air, cells were stained with ethidium bromide (SIGMA) adjusted to a final concentration of 20 μg/ml and observed under epifluorescent microscopy (OLYMPUS BH2‐RFCA, Olympus, Tokyo, Japan). More than 200 spermatozoa per slide were counted and classified into four groups according to the extent of the halo of sperm heads, namely, ‘without’, ‘small’, ‘medium’ or ‘large’ halos (Fig. 2). An inverse correlation was confirmed between halo size and DNA Breakage Detection‐Fluorescence In Situ Hybridization (DBD‐FISH) [16]. Sperm nuclei with small halos or without halos corresponded to extensive DNA fragmentation. The percentage of the number of sperm heads without halos and with small halos was used for a parameter of DNA fragmentation for SCD values in this study.

Figure 2.

Figure 2

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Photographs of spermatozoa in SCD test taken by epifluorescent microscopy. They were classified into four groups according to the extent of halos of sperm heads, namely, “without”, “small”, “medium” or “large” halos

Controlled ovarian stimulation and mode of insemination

To assess blastocyst development, those patients who had successfully obtained over five fertilized oocytes were included to rule out any complicating oocyte factors (n = 22). Controlled ovarian stimulation was conducted in all patients with the long protocol of GnRH analog reported elsewhere. ICSI was performed by Piezo‐ICSI alone [17].

Embryo culture

Embryos were cultured for 5 days sequentially in fertilization medium (Irvine), cleavage medium (Irvine) and blastocyst medium (Irvine). Fertilization was defined under a stereo microscope 16–18 h after insemination. Early embryos were transferred to blastocyst medium 3 days after insemination. After 120 h of culture, embryos reaching the early blastocyst stage with blastulation were regarded as appropriate blastocysts. The rate of blastocyst formation was expressed as the percentage (the number of blastocysts/the number of fertilized oocytes) × 100 (%)).

Statistical analysis

Results were shown as averages ± SD. Sperm chromatin structure, DNA fragmentation and the rate of blastocyst formation were examined by linear regression analysis. Statistical differences were considered to be significant at p < 0.05. Statistical analysis was performed by using SPSS software (12.0 J for Windows; SPSS Japan Inc., Tokyo).

Results

The mean age ± SD of the wives and husbands treated was 32.9 ± 3.2 years and 35.1 ± 4.6 years, respectively. The median duration of infertility, the number of retrieved oocytes and the fertilization rate was 6.4 ± 3.1 years, 12.2 ± 5.5 oocytes and 74.0 ± 16.1%, respectively.

A correlation between COMP values and embryo development as the rate of blastocyst formation was assessed (Fig. 3). There was a significant positive correlation between COMP values and the rate of blastocyst formation (r = 0.477, p = 0.025, n = 22). COMP values reflect the number of thiol bonds in sperm nuclear chromatin. It was supposed that spermatozoa with fewer thiol bonds related to better embryo development.

Figure 3.

Figure 3

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Relationship between sperm nuclear chromatin status (COMP values) and rate of blastocyst formation of human embryos derived from ICSI procedure (n = 22, r = 0.477, p = 0.025)

A correlation between SCD values and the rate of blastocyst formation was assessed by SCD test (Fig. 4). Although a significant correlation could not be observed in all 22 cases (r = 0.416, p = 0.050), there was a significant correlation in the 12 cases in which the femaleˈs age was lower than 35 years (r = 0.796, p < 0.001). Because SCD values were used for a parameter of DNA fragmentation, it was supposed that DNA fragmentation had a negative relationship with blastocyst formation, although more cases would be needed for confirmation.

Figure 4.

Figure 4

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Correlation between sperm nuclear DNA fragmentation (SCD values) and rate of blastocyst formation of human embryos obtained from cases under 35 years old (n = 12, r = 0.796, p < 0.001)

Finally, a correlation between these two values, COMP values and SCD values, was assessed (Fig. 5). Significant correlation could not be observed between these values (r = 0.224, p = 0.080). That is, there was no relationship between the number of disulfide bonds in sperm nuclear chromatin and the amount of DNA fragmentation.

Figure 5.

Figure 5

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Relationship between human sperm nuclear chromatin status (COMP values) and DNA fragmentation (SCD values) (n = 44, r = 0.224, p = 0.080)

Discussion

Sperm nuclei develop into species‐specific shapes during spermiogenesis within the seminiferous tubules. During this period, the major nuclear proteins evolve from histones, the somatic‐type proteins that exist until spermatid differentiation, into protamine [18]. By this alteration of nuclear proteins within nucleosomes, a sperm specific side‐by‐side DNA arrangement is accomplished. Paternal DNA is packaged tightly and genetic information is preserved safely. Passing through the epididymus, immature spermatozoa are modified in various molecular steps and become fertilizable. One of the changes in sperm nuclei occurring during this period is production of disulfide bonds within protamines, changing the thiol (–SH) moieties in cysteine, which is highly enriched in protamines. Resultant disulfide bonds provide the sperm nuclear stability [19]. With the exception of primates, in most mammals, the nuclear maturity of ejaculated spermatozoa is homogeneous and highly stable. The maturity of human ejaculated sperm nuclei, however, was demonstrated experimentally to be quite heterogeneous. AO fluorescence is an effective tool to demonstrate this heterogeneity [20]. Fluorescent patterns give an indicator of sperm nuclear maturity, reflecting the ability of fertilization in c‐IVF [5, 21]. Human sperm nuclei contain fewer protamine molecules (approximately 85%) than those of bulls, stallions, hamsters, and mice [22]. Most mammals express only one type of protamine (protamine‐1, P1) in sperm chromatin, however human and mouse produce an another type of protamine (protamine‐2, P2) that is deficient in cysteine residues. Consequently, the number of disulfide bonds is diminished in human sperm in comparison to species expressing P1 alone [23]. Abnormalities of proteolytic cleavage of the P2 precursor influence human sperm chromatin heterogeneity, producing latent infertility [24, 25]. In cases of idiopathic infertility, sperm chromatin study may reveal such aberrations if the conventional semen parameters defined to be normal by the World Health Organization (WHO) criterion [5, 26].

Some methods for detecting sperm nuclear DNA fragmentation have been reported, including toluidine blue staining [27], DNA breakage detection‐fluorescence in situ hybridization [28], in situ nick translation assays [29], comet assays [30], TUNEL assays [31] and sperm chromatin structure assay (SCSA®) [6]. SCSA® has been presented as a useful method for detecting sperm DNA fragmentation (DNA fragmentation index; DFI) by applying flow cytometry. AO staining can detect denaturation of sperm DNA by acid treatment following excitation with blue light (450–490 nm). In the case of completely matured sperm nuclei, which have many disulfide bonds, DNA is kept in double stranded form even if it is exposed to stresses such as acid. In this case, AO molecules are intercalated into double stranded DNA and green fluorescence (530 ± 30 nm) is emitted from nuclei. On the other hand, immature sperm nuclei with few disulfide bonds can be denatured to single strand form and AO molecules aggregates within the nuclei. Red fluorescence (>630 nm) can be observed at the same excitation [20] in immature sperm nuclei.

In this study, sperm chromatin was assessed by flow cytometry with AO. It was first reported by Evenson et al. [6] and our modifications provided several additional benefits. One of the modifications is using DTT for setting up the gate in advance. DTT is a disulfide reducing agent and it can change the emission from sperm heads effectively (Fig. 1b, c). In addition, aliquots were cryopreserved after treatment with NEM, a thiol‐blocking agent preventing the rearrangement of disulfide bonds in sperm nuclear chromatin. Preliminary experiments verified that treatment with DTT and NEM allowed more objective and precise assessment of sperm chromatin by flow cytometry using AO.

ICSI has provided improved outcomes for the couples with fertilization failure. In contrast to c‐IVF, chromatin status of the sperm nuclei used for ICSI is thought to be heterogeneous. Although some reports to the contrary can be found in the literature, the rate of blastocyst formation after ICSI is clinically lower than that seen in c‐IVF [7, 8, 9, 10, 11, 12]. In ICSI with ejaculated sperm, spermatozoa are selected by an operator under a magnification of only 200×–300×. It is assumed that the period during nuclear decondensation and paternal pronuclear formation may be altered after incorporation into ooplasm, depending on the chromatin status of the selected sperm head. Moreover, by‐passed acrosome reaction and sperm–egg fusion, and simultaneous injection of the sperm cytoplasmic membrane may have adverse effects on the synchronization between an oocyte and a spermatozoon [13, 14]. From this point of view, in the case of ICSI, chromatin status, especially thiol condition of sperm nuclei, can influence the process of fertilization and subsequent in vitro embryo development. It was suggested by using B6D2F1 mice that immature sperm nuclei with fewer disulfide bonds exhibited relatively faster decondensation and pronuclear formation after ICSI (data not shown), which is consistent the literature previously reported [32]. The ratio of inner cell mass (ICM), which was an index of embryo development, was also assessed during blastocyst formation. It was higher in fertilized ova injected with immature spermatozoa containing few disulfide bonds. That is, blastocysts injected with immature spermatozoa could develop faster in ICSI (data not shown).

Clinically, examination of in vitro embryonic development after ICSI did not reveal a significant correlation between the rate of blastocyst formation and the age of the patients, the duration of infertility, the number of retrieved oocytes or the conventional semen parameters, respectively in this study (data not shown).

Quality assessment of used spermatozoa may provide valuable information explaining why embryonic development is worse in ICSI than in c‐IVF. In this study, a correlation between sperm nuclear chromatin structure and blastocyst formation was assessed using COMP values obtained by flow cytometer in ICSI patients. There was a positive correlation (Fig. 3) between COMP values and the rate of blastocyst formation. It was suggested that embryo development proceeds more rapidly using spermatozoa with fewer disulfide bonds, as observed in our animal (B6D2F1 mice) experiments as mentioned above. In the case of ICSI, the process of male and female pronuclear formation could be dyssynchronized due to the structure of injected sperm nuclear protein, and the dyssynchrony might have an adverse effect on embryonic development.

Correlation between sperm DNA fragmentation and the rate of blastocyst formation was also assessed (Fig. 4) by SCD test. To exclude any oocyte factors, only those patients who had successfully obtained over five fertilized eggs were included. Although a significant correlation could not be observed in all 22 cases (r = 0.416, p = 0.050), there was a significant correlation in the 12 cases in which the femaleˈs age was lower than 35 years (r = 0.796, p < 0.001). It was pointed out that sperm DNA fragmentation had deteriorative effects on in vitro embryo development after ICSI, which was consistent with previous reports [33].

However, DNA fragmentation might occur easily in ejaculated spermatozoa with few disulfide bonds because of their structural weakness. So, the correlation of COMP values and SCD values were assessed (Fig. 5). From our study, a positive correlation could not be observed between COMP values and SCD values. It was suggested that severe damage of the paternal DNA was not related to the chromatin condition as a thiol status, therefore, the COMP values and the SCD values were thought to be independent parameters by which to assess sperm nuclear quality for developmental ability after an ICSI procedure.

From these results, it was suggested that injection of spermatozoon with fewer disulfide bonds and less DNA fragmentation could achieve better blastocyst formation in human ICSI. In our previous study, it was revealed that the nuclei of testicular sperm extracted from several mammalian species exhibited red fluorescence completely with AO [20]. That is, testicular sperm had no disulfide bonds within chromatin. Although we were apprehensive that DNA fragmentation might occur easily without disulfide bonds, DNA fragmentation was revealed to be less than 5% even in human testes [34]. It was suggested that testicular spermatozoa, which had no disulfide bonds and less DNA fragmentation in their chromatin, could improve embryonic development.

In conclusion, our study suggests that injection of spermatozoa with fewer disulfide bonds and less DNA fragmentation could achieve better blastocyst formation in human ICSI. Assessment of sperm chromatin and DNA fragmentation should help to determine whether repeated ICSI or testicular sperm extraction (TESE)‐ICSI is more appropriate [34]. Additional evidence will be needed to establish a practical assessment of human sperm quality to improve ICSI outcome.

Acknowledgments

This research was supported by Grants‐in‐Aid for Scientific Research from Japan Society for the Promotion of Science grant no. C‐15591771.

Contributor Information

Tomoko Takayama, Email: takayama@fmu.ac.jp.

Haruo Katayose, Email: katayose@iuhw.ac.jp.

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