Patrizia Grace Bianchi

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3.

Sperm decondensation during fertilisation in the mouse: presence of DNase I hypersensitive sites in situ and a putative role for topoisomerase II.

In this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 microM teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated.

Use of the guanine-cytosine (GC) specific fluorochrome, chromomycin A3, as an indicator of poor sperm morphology.

AbstractPurpose: We have previously postulated that the chromomycin A3(CMA3) fluorochrome allows an indirect visualization of sperm chromatin packaging quality and partially denatured sperm DNA. In this study we investigate the relationship between CMA3 positivity and sperm morphology. We also present data on the association between sperm morphology and the presence of endogenous nicks in sperm DNA. Methods: Semen samples were examined from 81 males of the couples who were consulting for infertility treatment. CMA3 fluorescence was assessed for all samples, while in 24 sperm samples we also examined for the presence of endogenous nicks in the sperm DNA. Results: When sperm morphology was less than 20% normal in a patient, the level of CMA3 fluorescence and presence of endogenous nicks were significantly higher than in patients with a higher incidence of morphologically normal sperm. Conclusions: CMA3 could be used as an adjunct to the assessment of morphology as an evaluation method for poor sperm. Its value in predicting fertilizing ability when using either SUZI or ICSI awaits to be answered.

Termination of Pregnancy and Women’s Sexuality

Objective: To determine the influence of termination of pregnancy (TOP) on women’s sexuality. Design: Prospective qualitative and quantitative study. Subjects: 103 women undergoing induced abortion by vacuum aspiration, interviewed 1–3 weeks before surgery and 6 months later. Results: After TOP, patients described symptoms of fatigue (39%), feelings of guilt (35%), sadness (34%) and anxiety (29%). Thirty-one percent of women presented at least one sexual dysfunction, 18% a decrease in sexual desire, 17% orgasmic disorders, 12% vaginal dryness and 11% dyspareunia. These sexual dysfunctions were correlated with anxiety and symptoms of depression following TOP. Six months after TOP, 57% of the women reported no change in their sexual satisfaction, 17% were ‘more satisfied’ and 7% ‘less satisfied’. Lessening of sexual satisfaction after TOP was correlated with diminished partner satisfaction (p < 0.00001), fatigue (p < 0.0009), feelings of guilt (p < 0.01), low frequency of sexual relations (p < 0.01) and anxiety over sexual relations (p < 0.02). Conclusions: Six months after TOP some women presented persisting sexual dysfunction. This sexual dysfunction may be explained essentially by the appearance of symptoms of anxiety and depression following TOP. When the quality of the relationship was satisfying, women could cope more easily with the appearance of a sexual dysfunction.

Human Cervical Mucus Can Act in Vitro as a Selective Barrier Against Spermatozoa Carrying Fragmented DNA and Chromatin Structural Abnormalities

AbstractPurpose: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. Methods: Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and “endogenous” nick translation. Results: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. Conclusions: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.