Chemotherapy drugs like cyclophosphamide (CPA) is an alkylating agent that can cause damage to the male germ cells and follicles of the gonads resulting in adverse reproductive problems and infertility. This study aims to investigate any potential to salvage the embryo from the toxic paternal exposure with the use of Nigella sativa extract (NSE) and thymoquinone (TQ). The survival and damage of embryos following fertilization from sperm exposed to CPA were studied in order to investigate the protective effects of NSE and TQ on male mice at 8 to 10 weeks of age according to the groupings as follows: i) control, ii) CPA only, iii) NSE only, iv) TQ only, v) CPA+NSE and vi) CPA+TQ, as well as supplementation of NSE (5mg/ml, 10mg/ml, and 15mg/ml) and TQ (1μM, 10μM and 100μM) in the culture media of developing embryo. Non-invasive embryo examination was based on simple methods of observation focused on morphology and dynamics of embryo development under the inverted microscope without fixation and staining. The level of embryo glucose uptake was then determined to evaluate the quality of the preimplantation embryos while quantitative polymerase chain reaction (Q-PCR) and immunofluorescence methods were employed to investigate the tendency of the embryos whether to survive or undergo apoptosis. In addition, the effects of NSE and TQ supplementation in the in vitro fertilization (IVF) culture media were also investigated. The results obtained from the preliminary study involving Balb/c has shown that the introduction to NSE and TQ have potential protective effects on the development of the embryo in vitro with a significant (p<0.05) in the percentage of motile sperm, number of fragmented DNA, sperm morphology, sperm head abnormalities and fertilization rate. NSE and TQ were also seen to improve the embryo quality in terms of embryo grading based on the fragmentation and structure of blastomere. The experiment using Swiss bred mice strain (ICR) has proved that treatment with NSE and TQ has a significant positive impact (p<0.05) on sperm motility, reduction in the number of abnormal sperm and percentage of sperm head abnormalities. Study on the embryo metabolites, indicates that there is no significant difference (p>0.05) in the level of glucose uptake in the culture media between the groups. In addition, molecular study which investigated the presence of five genes important in embryo development (Bcl-xL, BAX, SOX2, Oct4, and CD29), revealed that there are no significant differences (p>0.05) in the levels of gene expression. The molecular findings were confirmed with immunofluorescence staining of the embryo for the detection of Bcl-xL, BAX, and Oct4. NSE and TQ supplementation in the culture media showed no effect on the embryo quality. Results from the in vivo experiments demonstrated the potential of NSE and TQ in the paternal protection from the alkylating effects of CPA to allow for the normal development of the embryo.

Advances in the treatment of cancer have allowed adolescent patients to become long-term survivors able to lead normal lives. However, the concern about the effects of anticancer drugs on fertility has led to many efforts to preserve germ cells of these patients. This study focuses on ways to assess the effectiveness of Nigella sativa and its active compound, thymoquinone, in lowering chemotherapeutic-associated toxicity of cyclophosphamide on the ovaries and testes of Balb/c mice. Histological and morphological effects on the germ cells were examined via microscopy techniques and possible fragmentations of sperm DNA was assessed using the COMET Assay. The RNA expression of OGG1 and FGF2 in the testicular cells was quantitatively analysed using the real-time PCR followed by quantification of the stages of fertilisation and embryo division using the inverted microscope. Supplementation of N. Sativa oil and thymoquinone exhibited noticeable protective effects on the histology and morphometry of the ovaries and seminiferous tubules as well as being effective in reducing the total DNA fragmentation in spermatozoa. N. Sativa extract reduced the percentage of abnormal sperm head post to cyclophosphamide treatment and preserved the normal chromatin condensation indicative of protection against sperm DNA alteration. The expressions of DNA repair and fibroblast growth factor genes were also shown to increase suggestive of possible reduction in mutagenic modifications. Thymoquinone supplementation increased the implantation and fertilisation rates, pregnancy outcome as well as preserved fair quality embryos following paternal and maternal exposures to cyclophosphamide. N. Sativa and thymoquinone are both suitable exogenous agents that offer viable chemoprotective potential against toxicity induced by cyclophosphamide. This study is part of an effort towards improving interventions to preserve fertility and to assist in the development of techniques in achieving favourable reproductive outcomes for adults who survived childhood cancer following chemotherapy.