Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg2+, which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat.

Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients.

Chromomycin A2 induces autophagy in melanoma cells.

The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.

The crucial role of divalent metal ions in the DNA-acting efficacy and inhibition of the transcription of dimeric chromomycin A3.

Chromomycin A3 (Chro) is capable of forming a stable dimeric complex via chelation with Ni(II), Fe(II) and Co(II). According to the circular dichroism study, the dimer conformations are significantly different among the Fe(II)-, Co(II)-, and Ni(II)-containing dimeric Chro complexes; however, the dimer conformations were preserved at high temperatures. Furthermore, we conducted a systematic study to determine the effects of these divalent metal ions on the DNA-acting efficacy of dimeric Chro, including its DNA-binding affinity, DNA stabilization capacity, DNA cleavage activity, and the inhibition of transcription both in vitro and within cells. Kinetic analyses using surface plasmon resonance (SPR) showed that Ni(II)(Chro)(2) exhibited the highest K(a) with a value of 1.26 × 10(7) M(-1), which is approximately 1.6- and 3.7-fold higher than the K(a) values obtained for Co(II)(Chro)(2) and Fe(II)(Chro)(2), respectively. The T(m) and ΔG values for the DNA duplex increased after the addition of drug complexes in the following order: Ni(II)(Chro)(2)>Co(II)(Chro)(2)>Fe(II)(Chro)(2). In the DNA integrity assays, the DNA cleavage rate of Co(II)(Chro)(2) (1.2 × 10(-3) s(-1)) is higher than those of Fe(II)(Chro)(2) and Ni(II)(Chro)(2), which were calculated to be 1 × 10(-4) and 3.1 × 10(-4) s(-1), respectively. Consistent with the SPR and UV melting results, Ni(II)(Chro)(2) possesses the highest inhibitory effect on in vitro transcription and c-myc transcription within cells compared to Co(II)(Chro)(2) and Fe(II)(Chro)(2). By comparing the cytotoxicity among Co(II)(Chro)(2), Fe(II)(Chro)(2), and Ni(II)(Chro)(2) to several cancer cell lines, our studies concluded that Ni(II)(Chro)(2) displayed more potential antitumor activities than Co(II)(Chro)(2) and Fe(II)(Chro)(2) did due to its higher DNA-acting efficacy. Changes to the divalent metal ions in the dimeric Chro complexes have been correlated with improved anticancer profiles. The availability of new metal derivatives of Chro may introduce new possibilities for exploiting the unique properties of this class of compounds for therapeutic applications.

Comparative cytogenetic mapping between the lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.).

The common bean (Phaseolus vulgaris) and lima bean (P. lunatus) are among the most important legumes in terms of direct human consumption. The present work establishes a comparative cytogenetic map of P. lunatus, using previously mapped markers from P. vulgaris, in association with analyses of heterochromatin distribution using the fluorochromes chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) and localization of the 5S and 45S ribosomal DNA (rDNA) probes. Seven BACs selected from different common bean chromosomes demonstrated a repetitive pericentromeric pattern corresponding to the heterochromatic regions revealed by CMA/DAPI and could not be mapped. The subtelomeric repetitive pattern observed for BAC 63H6 in most of the chromosome ends of common bean was not detected in lima bean, indicating lack of conservation of this subtelomeric repeat. All chromosomes could be identified and 16 single-copy clones were mapped. These results showed a significant conservation of synteny between species, although change in centromere position suggested the occurrence of pericentric inversions on chromosomes 2, 9 and 10. The low number of structural rearrangements reflects the karyotypic stability of the genus.

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic.

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu(2+). CHR forms a high affinity 2:1 (CHR:Cu(2+)) complex with dissociation constant of 0.08 × 10(-10) M(2) at 25°C, pH 8.0. The affinity of CHR for Cu(2+) is higher than those for Mg(2+) and Zn(2+) reported earlier from our laboratory. CHR binds preferentially to Cu(2+) in presence of equimolar amount of Zn(2+). Complex formation between CHR and Cu(2+) is an entropy driven endothermic process. Difference between calorimetric and van't Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)(2):Cu(2+)] complex assumes a structure different from either of the Mg(2+) and Zn(2+) complex reported earlier. Both [(CHR)(2):Mg(2+)] and [(CHR)(2):Zn(2+)] complexes are known to bind DNA. In contrast, [(CHR)(2):Cu(2+)] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5'-CCGGCGCCGG-3'). In order to interact with double helical DNA, the (antibiotic)(2) : metal (Mg(2+) and Zn(2+)) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu(2+) complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg(2+) and Zn(2+). The results also indicate that CHR has a potential for chelation therapy in Cu(2+) accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.

Karyotype diversity of four species of the incertae sedis group (Characidae) from different hydrographic basins: analysis of AgNORs, CMA3 and 18S rDNA.

A large number of genera in the tropical fish family Characidae are incertae sedis. Cytogenetic analysis was made of four of these species: Astyanax eigenmanniorum, Deuterodon stigmaturus, Hyphessobrycon luetkenii, and H. anisitsi, collected from various hydrographic basins: hydrographic system from Laguna dos Patos/RS, Tramandaí basin/RS and Tibagi River basin/PR. The first two species were collected in their type locality in the State of Rio Grande do Sul. The 2n = 48 karyotype was observed only in A. eigenmanniorum, while the other species had 2n = 50 chromosomes, with different karyotypic formulas. There was weak heterochromatin staining in the pericentromeric region of A. eigenmanniorum, D. stigmaturus and H. luetkenni chromosomes. In H. anisitsi, heterochromatin appeared to be more abundant and distributed in the pericentromeric and terminal regions of the chromosomes; three pairs showed more evident heterochromatic blocks. There were multiple Ag-NORs in all populations, visualized by FISH with an 18S rDNA probe. While D. stigmaturus and H. luetkenii had conserved AgNOR, CMA3 and 18S rDNA sites, the other two species showed intra- and interindividual variation at these sites. The karyotype variability was high, as is common in this group of fish. Different species arising from isolated hydrographic basins maintain an elevated level of karyotype differentiation, mainly with respect to chromosome structure, heterochromatin distribution and rDNA localization. This is the first report with cytogenetic data for D. stigmaturus and H. luetkenii.

Quantitative expression of phospholipase C zeta, as an index to assess fertilization potential of a semen sample.

BACKGROUND: Failed fertilization post-ICSI has been mainly attributed to the sperm's inability to induce oocyte activation. Phospholipase C zeta (PLCζ) is considered to be one of the factors for the induction of oocyte activation. The aim of this study was to quantitatively assess the expression of PLCζ in globozoospermic men or those with previously low or failed fertilization in comparison with fertile men or those with high fertilization potential. In addition, the relationship between expression of PLCζ and that of other sperm markers was evaluated. METHODS: Real-time PCR was carried out to evaluate relative expression of PLCζ mRNA. Chromatin maturity and acrosin activity were assessed by CMA3 staining and a colorimetric method. RESULTS: The expression of PLCζ was significantly lower in globozoospermic men (P< 0.01, n= 8) or individuals with previously low or failed fertilization (P< 0.01, n= 36) in comparison to fertile men (n= 24). In addition, a significant difference was observed between globozoospermic (P< 0.01) and individuals with previously low or failed fertilization (P= 0.003) in comparison to high fertilization individuals (n= 17). Expression of PLCζ was not correlated with either chromatin maturity or acrosin activity. However, a significant correlation was observed between the percentage of fertilization and relative expression of PLCζ (r= 0.4, P< 0.01). CONCLUSION: In this study, for the first time, we have shown that assessment of relative expression of PLCζ may provide a useful marker for the ability of sperm to induce oocyte activation after ICSI.

Genomic content and new insights on the origin of the B chromosome of the cichlid fish Astatotilapia latifasciata.

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1­2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C0t - 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.

Sequence-based high resolution chromosomal comparative genomic hybridization (CGH).

We aimed to devise an appropriate method to directly link the fluorescence profile of chromosomal copy number alterations detected by chromosomal comparative genomic hybridization (cCGH) or any other hybridization or staining information with the genome sequence data. Our goal was to establish an internal anchoring system that could facilitate profile alignment and thus increase the resolution of cCGH. We were able to achieve the alignment of chromosomes with gene mapping data by superimposition of (a) the fluorescence intensity pattern of a sequence-specific fluorochrome (GGCC binding specificity), (b) the cCGH fluorescence intensity profile of individual chromosomes, and (c) the GGCC motif density profile extracted from a genome sequence database. The adjustment of these three pieces of information allowed us to precisely localize, in cytobands and mega base pairs (Mb), regions of genomic alterations such as gene amplifications, gains, or losses. The combined visualization of sequence information and cCGH data together with application of the Warp tool, presented here, considerably improves the cCGH accuracy by increasing its resolution from 10 to 20 Mb to less than 2 Mb.

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A(3).

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co(II)(Chro)(2) complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6mM concentration of spermine completely abolished the DNA-binding activity of Fe(II)(Chro)(2) and Cu(II)(Chro)(2) and interfered with the associative binding of Co(II)(Chro)(2) complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2mM) promoted DNA strand cleavage by Cu(II)(Chro)(2), whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co(II)(Chro)(2) or Fe(II)(Chro)(2). Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe(II)(Chro)(2). Despite the fact that Cu(II)(Chro)(2) and Fe(II)(Chro)(2) demonstrated lower DNA-binding activity than Co(II)(Chro)(2) in the absence of spermine, while Cu(II)(Chro)(2) and Fe(II)(Chro)(2) exhibited greater cytoxicity against HepG2 cells than Co(II)(Chro)(2), possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.

Effects of failed oocyte activation and sperm protamine deficiency on fertilization post-ICSI.

Sperm premature chromosomal condensation (PCC) has been associated with failed fertilization. Previous studies suggest that protamine deficiency or failed oocyte activation may make spermatozoa prone to PCC. However, it is not clear which of these two factors has a more profound effect on fertilization failure. In order to distinguish between these two phenomena, oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) were artificially activated and the association between protamine deficiency and PCC was evaluated in the remaining oocytes that failed to fertilize. The results of this study reveal that after artificial activation, fertilization rate post-ICSI increased from 59.95 to 87.7% and PCC spermatozoa appeared to be present in over 50% of the remaining oocytes that failed to fertilize. The percentage of sperm PCC was significantly higher in protamine deficient samples, thus suggesting that after failed oocyte activation, sperm PCC induced by protamine deficiency may be considered as an alternative cause of failed fertilization post-ICSI. Furthermore, the results of this study did not show any correlation between pronuclei size asynchrony and protamine deficiency.

Exposure of male rats to cyclophosphamide alters the chromatin structure and basic proteome in spermatozoa.

BACKGROUND: The formation of mature sperm involves the expression of numerous proteins during spermiogenesis and the replacement of histones with protamines to package the genome. Exposure to cyclophosphamide (CPA), an anticancer alkylating agent, during spermiogenesis may disrupt chromatin condensation with adverse consequences to the offspring. METHODS: Adult male rats were given CPA in one of two schedules: (i) subchronic, 4 days - day 1 (100 mg kg(-1)) and days 2-4 (50 mg kg(-1) per day) or (ii) chronic - daily (6.0 mg kg(-1) per day). Animals were euthanized on days 14, 21 or 28. RESULTS: The effects of CPA on epididymal sperm chromatin structure were germ-cell-phase specific; mid-spermiogenic spermatids were most sensitive. The acridine orange DNA denaturation assay showed significant increases in susceptibility to denaturation (P < 0.01). Chromatin packaging assessment revealed 1,4-dithiothreitol-dependent chromomycin A3 DNA binding and less condensed, protamine-deficient sperm; the total thiol (P < 0.001) and protamine contents (P < 0.01), measured using monobromobimane and the HUP1N protamine 1 antibody, respectively, were reduced. The sperm basic proteome was also altered; proteins that were identified are involved in events during spermiogenesis and fertilization. CONCLUSIONS: Paternal exposure to CPA alters sperm chromatin structure, as well as the composition of sperm head basic proteins. We speculate that these changes underlie effects on fertilization and embryo development.

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin.

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.

ICSI outcome is not associated with the incidence of spermatozoa with abnormal chromatin condensation.

BACKGROUND: The condensation of sperm chromatin during spermiogenesis and epididymal transport is of essential importance for fertilization. The main purpose of this study was to examine whether abnormalities of sperm nuclear condensation can influence the outcome of intracytoplasmic sperm injection (ICSI) cycles. MATERIALS AND METHODS: Semen samples from 154 ICSI cycles were studied. Before semen preparation for ICSI, basic semen analysis was performed and a small portion from each sample was fixed. The condensation of sperm nuclear chromatin was evaluated with chromomycin A3 under a fluorescence microscope. RESULTS: The incidence of spermatozoa with abnormal chromatin condensation was positively correlated with sperm concentration (p = 0.020565), but was not correlated with other semen parameters such as morphology and motility. Abnormal chromatin condensation was also not correlated with fertilization rate, cumulative embryo score or pregnancy rate. CONCLUSION: The above results indicate that ICSI outcome is not influenced by the incidence of spermatozoa with abnormal chromatin condensation.

New DNA binding ligands as a model of chromomycin A3.

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this study, new DNA-binding ligands were designed to mimic Chromomycin A(3) (CRA(3)), which contains a hydroxylated tetrahydroanthracene chromophore substituted with di and trisaccharides. The trisaccharide part of CRA(3) that is supposed to contribute to form the Mg(2+)-coordinated dimer was expected to be mimicked by a simple alkyl group attached to the chromophore part as new model compounds. The present study has successfully demonstrated that the new ligands form Mg(2+)-coordinated dimer complexes to exhibit DNA-binding affinity.

Crystal structure of the [Mg2+-(chromomycin A3)2]-d(TTGGCCAA)2 complex reveals GGCC binding specificity of the drug dimer chelated by a metal ion.

The anticancer antibiotic chromomycin A3 (Chro) is a DNA minor groove binding drug belonging to the aureolic family. Chro likely exerts its activity by interfering with replication and transcription. Chro forms a dimer, mediated by a divalent metal ion, which binds to G/C-rich DNA. Herein we report the first crystal structure of Chro bound to d(TTG GCCAA)2 DNA duplex solved by multiwavelength anomalous diffraction (MAD) based on the chelated Co3+ ion. The structure of the Mg2+ complex was subsequently refined at 2.15 A resolution, which revealed two complexes of metal-coordinated dimers of Chro bound to the octamer DNA duplex in the asymmetric unit. The metal ion is octahedrally coordinated to the O1 and O9 oxygen atoms of the chromophore (CPH), and two water molecules act as the fifth and sixth ligands. The two coordinated water molecules are hydrogen bonded to O2 atoms of C5 and C13 bases. The Chro dimer binds at and significantly widens the minor groove of the GGCC sequence. The long axis of each chromophore lies along and stacks over the sugar-phosphate backbone with the two attached saccharide moieties (rings A/B and C/D/E) wrapping across the minor groove. DNA is kinked by 30 degrees and 36 degrees in the two complexes, respectively. Six G-specific hydrogen bonds between Chro and DNA provide the GGCC sequence specificity. Interestingly, DNA in concert with Chro appears to act as an effective template to catalyze the deamination of Co(NH3)6(3+), as shown by circular dichroism and crystal structure data. Our results present useful structural information for designing new anticancer drug derivatives in the future.

Effect of protamine-2 deficiency on ICSI outcome.

During spermiogenesis, histones are replaced by protamines (P1 and P2), resulting in sperm chromatin condensation followed by a halt to gene expression in haploid spermatids and spermatozoa. As a consequence, protamine deficiency and aberrant P1/P2 ratio have a profound effect on both fertilization and embryo development. However, reports on the effect of the P1/P2 ratio on fertilization and embryo development after intracytoplasmic sperm injection (ICSI) are contradictory between human and animal studies. The question that still remains to be elucidated is which type of protamine deficiency is most common among protamine deficient samples. The present study has a direct bearing on this issue investigating the correlation of the P1/P2 ratio with protamine deficiency, fertilization, embryo quality and embryo development in ICSI patients. This study was carried out on 71 patients. Chromomycin A3 (CMA3) staining was used to determine protamine deficiency. Since this procedure does not indicate the type of protamine deficiency, the P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel electrophoresis and analysis of protein bands with software. Polyclonal anti-P1 and anti-P2 antibodies were used to confirm P1 and P2 presence. Results show a negative significant correlation of fertilization rate with protamine deficiency and P1/P2 ratio. No significant correlation was observed between protamine deficiency and P1/P2 ratio. Therefore, it can be concluded that altered P1/P2 ratio effects fertilization rate and embryo quality which subsequently may affect implantation and pregnancy outcome.