Epigenetic changes in preimplantation embryos subjected to laser manipulation.

The advantage of using laser for assisted hatching in routine assisted reproductive technology (ART) practice is debatable. Recently, it has been shown that laser-manipulated mouse embryos had compromised genetic integrity. However, the impact of laser-assisted hatching (LAH) on the epigenetic integrity of the preimplantation embryos is not elucidated so far. Since continuous thermal stress on embryos was found to lower mRNA levels of de novo (bovine) methyl transferases in embryos, we hypothesize that thermal energy induced during LAH may alter the epigenetic signature through abnormal de novo methyl transferases (Dnmts) levels. Thus, using mouse model, we made an attempt to look into the expression of Dnmt3a and Dnmt3b in laser-manipulated embryos and their effects on global methylation. This experimental prospective study used mouse embryos from varying developmental stages (2-cell, 6-8-cell, and blastocyst) which were subjected to LAH using a 1480-nm diode laser. Two pulses of 350 µs frequency were applied to breach the zona pellucida, and then, embryos were assessed for the expression of two de novo methyl transferases (Dnmt3a and Dnmt3b) and LINE-1 (long interspersed element-1) methylation when LAH embryos developed to blastocyst stage. Results from this study have shown that blastocysts subjected to LAH at two-cell stage had significantly lower mRNA transcripts of Dnmt3a (P < 0.01) and Dnmt3b (P < 0.05) whereas LAH at six- to eight-cell and blastocyst stages did not affect the mRNA level significantly. On the other hand, LINE-1 methylation did not change significantly between LAH and control group in all the stages studied. These results suggest that two-cell-stage laser manipulation of embryos changes the mRNA level of Dnmts without affecting the global DNA methylation.

Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos.

Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs.

Ethanolic extract of Moringa oleifera Lam. leaves protect the pre-pubertal spermatogonial cells from cyclophosphamide-induced damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera Lam. is widely cultivated in Asian and African countries for its medicinal and dietary significance. The leaves are highly nutritious and are known to possess various biological activities. MATERIALS AND METHODS: Pre-pubertal Swiss albino male mice were injected with single dose of cyclophosphamide (CP, 200mg/kg body weight) or ethanolic extract of Moringa oleifera leaves (MOE, 100mg/kg body weight) intraperitoneally. In combination group, MOE was administered 24h prior to CP injection. RESULTS: CP induced a significant decrease in testicular weight (p<0.01) and depletion of germ cells (p<0.001) and higher level of DNA damage (p<0.001) compared to control. The expression of P53, Bax, Cytochrome C (Cyt C) was increased while there was a decrease in the expression of Bcl2, c-Kit and Oct4. Administration of MOE 24h prior to CP treatment ameliorated the depletion (p<0.001), DNA damage (p<0.001) and apoptosis (p<0.01) of germ cells induced by CP. The mitigating effect of MOE appears to be mediated by up-regulating the expression of c-Kit and Oct4 transcripts in P53-independent manner. CONCLUSION: MOE protects the spermatogonial cells from CP-induced damage by modulating the apoptotic response elicited by CP and therefore can be considered as an efficient method of male fertility preservation.

Laser-assisted hatching of cleavage-stage embryos impairs developmental potential and increases DNA damage in blastocysts.

This study aims to investigate the influence of two- (day 2) and six-to-eight-cell-stage (day 3) laser-assisted hatchings on the developmental potential and genetic integrity of the embryos. In this prospective experimental study, two- and six-to-eight-cell-stage mouse embryos were subjected to laser hatching using 1,480 nm diode laser, and then assessed for the developmental potential and DNA integrity in blastocysts. Similarly, four-cell-stage human embryos from 20 patients were also subjected to laser hatching, and then assessed for the developmental competence. Laser-assisted hatching in mouse embryos significantly enhanced the blastocyst hatching potential on day 4.5 (P < 0.0001). However, a significant decline in blastocyst total cell number (TCN) was observed in six-to-eight-cell-stage laser-hatched embryos (P < 0.001). Conversely, no significant difference in TCN was observed between laser-hatched and unhatched human four-cell-stage embryos after 24 h. Attempt to understand the genetic integrity in laser-hatched mouse blastocysts revealed significantly higher labeling index when hatching was done at two- (P < 0.01) and six-to-eight-cell stage (P < 0.05). DNA damage induced by the laser manipulation may affect implantation and postimplantation developmental potential of the embryos. However, further studies are required to elucidate the impact of laser-induced DNA damage on the reproductive outcome.

Oocytes recovered after ovarian tissue slow freezing have impaired H2AX phosphorylation and functional competence.

It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze-thawing. Using a mouse model, here we have investigated the functional competence and phosphorylation of H2AX (γ-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with γ-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P<0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P<0.05) and control (P<0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P<0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome.