Nicoletta Tarozzi

Nature of DNA Damage in Ejaculated Human Spermatozoa and the Possible Involvement of Apoptosis

Abstract Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.

Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis.

The mechanisms responsible for producing abnormal spermatozoa in the ejaculate are relatively unknown. Numerous studies have now shown the presence of nuclear DNA strand breaks in human ejaculated spermatozoa and the abnormal persistence of apoptotic marker proteins. The reason why human spermatozoa, in particular from men with abnormal semen parameters, possess these abnormalities is still not clear. Two processes that have been linked to the presence of nuclear DNA strand breaks in spermatozoa are anomalies in apoptosis during spermatogenesis or problems in the replacement of histones with protamines during spermiogenesis. Understanding the mechanisms responsible for producing abnormal spermatozoa in the human will improve knowledge about certain causes of male infertility.

Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

Cryopreservation of human spermatozoa—introduced in the 1960s—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART): appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

Anomalies in sperm chromatin packaging: implications for assisted reproduction techniques

Sperm protamine deficiency and DNA damage were analysed employing chromomycin A(3) (CMA(3)) staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, respectively, in 132 patients (82 IVF, 50 intracytoplasmic sperm injection [ICSI]). The antioxidant ability of seminal plasma was analysed in 10 men, using the total oxidant scavenging capacity assay. A significant negative correlation was found between abnormal protamination and sperm parameters, including sperm DNA fragmentation (P < 0.01). A close relationship was found between sperm protamination and fertilization and pregnancy only in IVF (P = 0.004 and P < 0.04, respectively); in ICSI there was a correlation between DNA fragmentation and pregnancy (P = 0.031). Finally, there was a negative correlation between chromatin under-protamination and the antioxidant ability of seminal plasma (P < 0.01). Results of this study underline that, despite sperm abnormal protamination and DNA fragmentation being positively correlated, they affect the reproductive outcome in different ways: in particular there was good prognostic value for CMA(3) analysis only in IVF, whereas DNA fragmentation analysis was prognostic only for ICSI outcome. Data are also provided to support the idea of a relationship between defective antioxidant system activity and impairment of chromatin packaging.

Sperm–hyaluronan-binding assay: clinical value in conventional IVF under Italian law

Hyaluronan has recently been employed in the development of a commercial diagnostic kit for assessing sperm maturity, the so-called sperm-hyaluronan-binding assay (HBA). The aim of this study was to evaluate the usefulness of this test, in addition to routine semen analysis, to identify patients with poor reproductive prognosis in conventional IVF. Furthermore, the ability of hyaluronan to select spermatozoa with low DNA fragmentation was investigated. A total of 60 IVF patients were analysed with regard to reproductive outcome, sperm parameters, HBA score and sperm DNA fragmentation. The DNA fragmentation analysis was performed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay on the total sperm population and on the hyaluronan-bound spermatozoa obtained from the same samples. No relationship between hyaluronan binding and fertilization, cleavage, good-quality embryos, implantation, clinical pregnancy, miscarriages and biochemical pregnancy rates was found. Otherwise, correlations between spermatozoa hyaluronan binding and morphology (P < 0.01) and a significant difference between DNA fragmentation of the total sperm population and DNA fragmentation of the hyaluronan-bound spermatozoa (P = 0.029) were found. The results underline the ability of hyaluronan to select spermatozoa with higher DNA integrity and morphology. Nevertheless, the clinical value of the HBA in the management of male infertility seems to be limited.

Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing

OBJECTIVE To determine the pregnancy outcome potential of mosaic embryos, detected by means of preimplantation genetic screening (PGS) with the use of next-generation sequencing (NGS). DESIGN Retrospective study. SETTING Genetics laboratories. PATIENT(S) PGS cycles during which either mosaic or euploid embryos were replaced. INTERVENTION(S) Blastocysts were biopsied and processed with the use of NGS, followed by frozen embryo transfer. Trophectoderm (TE) biopsies were classified as mosaic if they had 20%-80% abnormal cells. MAIN OUTCOME MEASURE(S) Implantation, miscarriage rates, and ongoing implantation rates (OIRs) were compared between euploid and types of mosaic blastocysts. RESULT(S) Complex mosaic embryos had a significantly lower OIR (10%) than aneuploidy mosaic (50%), double aneuploidy mosaic (45%), and segmental mosaic (41%). There was a tendency for mosaics with 40%-80% abnormal cells to have a lower OIR than those with <40% (22% vs. 56%). However, few embryos (n = 34) with a mosaic error in 40%-80% of the TE sample were replaced. There was no difference between monosomic and trisomic mosaics or between entire chromosome mosaicism or segmental mosaicism. Implantation rates were significantly higher (70% vs. 53%), miscarriage rates lower (10% vs. 25%), and OIRs higher (63% vs. 40%) after euploid embryo transfer than after mosaic embryo transfer. CONCLUSION(S) Forty-one percent of mosaic embryos produced an ongoing implantation. Complex mosaic blastocysts had a lower OIR than other mosaics. Mosaic monosomies performed as well as mosaic trisomies and mosaic segmental aneuploidies. The results suggest that embryos with >40% abnormal cells and those with multiple mosaic abnormalities (chaotic mosaics) are likely to have lower OIRs and should be given low transfer priority.

Vaginal gel versus intramuscular progesterone for luteal phase supplementation: a prospective randomized trial

The aim of this randomized study was to compare the efficacy of intramuscular progesterone (IMP) and progesterone in vaginal gel (VGP) at two different doses for luteal support in IVF. A total of 412 patients, aged between 28 and 37 years, were randomized into three groups. The day after oocyte retrieval each patient began supplementation with one of the following: IMP 50 mg daily (150 patients), VGP 90 mg once daily (143 patients), or VPG 90 mg twice daily (148 patients). No significant difference was found between the three groups in any of the endpoints. The rate of positive beta-human chorionic gonadotrophin per transfer was 38.4% with IMP, 35.0% with VPG once daily and 43.1% with VPG twice daily. Clinical pregnancy rate per transfer and implantation rate were 32.6% and 19.6% with IMP, 26.3% and 16.4% with one dose of VGP, and 37.2% and 21.1% with two doses of VGP. Live birth rate per transfer was 26.1%, 23.4% and 29.9%, respectively. Progesterone vaginal gel can be successfully used as an alternative to intramuscular progesterone for luteal support in IVF. One daily dose appears sufficient to induce clinical pregnancies and live births at a rate comparable to intramuscular supplementation.

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.

Annexin V magnetic-activated cell sorting versus swim-up for the selection of human sperm in ART: is the new approach better then the traditional one?

PurposeTo investigate whether the sperm fertilizing potential can be improved by selecting a non-apoptotic fraction using magnetic activated cell sorting (MACS), and to compare the results with the conventional swim-up method.MethodsTwenty five male patients attending the andrology laboratory for sperm DNA fragmentation analysis. The sperm were prepared by density gradient centrifugation (DGC) and subsequently divided into three aliquots. The first was further separated into Annexin V-negative (non-apoptotic) fraction using MACS, the second was further processed by swim-up, while the third was left unseparated as a control. The impact of the combination of DGC with the two sperm preparation techniques on sperm quality was evaluated by comparing ‘rapid progressive’ motility, normal morphology according to Tygerberg’s strict criteria and DNA integrity (by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling [TUNEL]) for each aliquot.ResultsSperm preparation that combines DGC with conventional swim-up method can provide sperm of higher quality in terms of motility, morphology and extent of DNA fragmentation compared to the Annexin V-negative (non-apoptotic) fraction derived from the combination of DGC with MACS.ConclusionsIntegrating MACS as a part of sperm preparation technique will not improve sperm fertilizing potential to the same extent as the traditional swim-up separation procedure.

Sperm DNA fragmentation testing in male infertility work-up: are we ready?

We read with interest the guideline proposed by Agarwal and colleagues (1) on clinical utility of sperm DNA fragmentation (SDF) testing. The Authors stated that, despite the clear association between SDF and male fertility, the clinical implication of SDF is poorly understood. So, the aim of the guideline was to underline the actual indications of SDF testing and also to explain the management of patients with increased SDF. To achieve this purpose, the Authors examined original and review articles concerning the significance of SDF testing and arranged their manuscript into two main sections: in the first part they described the current tests for SDF evaluation, underlining their basic principles as well as the main advantages and disadvantages; in the second part they performed an evidence-based analysis of the utility of SDF tests under specific clinical scenarios, commonly found by urologists and reproductive specialists. In particular, in the clinical scenario #1 varicocele was analyzed: Agarwal and colleagues stated that SDF testing may allow to better select varicocelectomy candidates among patients with clinical varicocele and borderline to normal sperm characteristics. In clinical scenario #2 the authors considered the unexplained infertility, suggesting performing SDF testing in couples with recurrent spontaneous abortion (RSA) or before starting intrauterine insemination (IUI). Clinical scenario #3 described the relationship between SDF and assisted reproduction techniques (ART), recommending the SDF analysis in patients with recurrent ART failures. Finally, in clinical scenario #4 the influence of lifestyle risk factors on male fertility was considered, suggesting offering SDF testing to infertile men with evidence of this kind of exposure, in order to underline an eventual sperm DNA damage and/or to monitor the patient’s response to treatment.