Association between total globozoospermia and sperm chromatin defects

Globozoospermia is a severe sperm morphological anomaly leading to primary infertility and low fertilisation following intracytoplasmic sperm injection (ICSI). This phenotype is observed in less than 0.1% of infertile men and is determined by small, round‐headed spermatozoa with absence of an acrosomal cap, acrosome protease and also cytoskeletal proteins. Failure of oocyte activation is considered as the main cause of fertilisation failure in these individuals post‐ICSI. Therefore, artificial oocyte activation (AOA) along with ICSI is commonly implemented. However, based on previous report, fertilisation rate remains low despite implementation of ICSI‐AOA. Therefore, other mechanisms like sperm chromatin packaging and DNA fragmentation may account for low fertilisation and development post‐ICSI‐AOA. Therefore, this study aims to assess and compare the degree of sperm protamine deficiency and DNA fragmentation in large population of infertile men with total globozoospermia (30 globozoospermic men presenting with 100% round‐headed spermatozoa) with 22 fertile individuals using chromomycin A3 and TUNEL assay respectively. Results clearly show that mean of sperm concentration and percentage of sperm motility were significantly lower, while percentage of sperm abnormal morphology, protamine‐deficient and DNA‐fragmented spermatozoa were significantly higher in infertile men with globozoospermia compared to fertile men. Therefore, increased sperm DNA damage in globozoospermia is likely related to defective DNA compaction and antioxidant therapy before ICSI‐AOA could be recommended as an appropriate option before ICSI‐AOA.     

Relationship between human spermatozoa–hyaluronan‐binding assay,conventional semen parameters and fertilisation rates in intracytoplasmic spermatozoa injection

Selecting the best spermatozoa for intracytoplasmic spermatozoa injection (ICSI) has recently been a topic of great interest among embryologists. The study aimed to evaluate the relationship between the spermatozoa‐hyaluronan‐binding assay (HBA), routine semen analysis results and fertilisation rates as recorded during conventional ICSI therapy. Ninety‐one patients undergoing conventional ICSI treatment in the Medfem Fertility Clinic in Johannesburg (South Africa) were included in the study. A total of 797 oocytes were injected of which 457 oocytes fertilised (57.3%, range 0–100%). None of the semen parameters correlated with the fertilisation rates (Table 2). HBA scores, however, revealed a highly significant association (p ≤ 0.0001) with the fertilisation rates. The HBA scores also correlated significantly with the biochemical pregnancy values (Spearman r = 0.24, P = 0.02, 95% CI 0.039–0.43); however, the HBA scores did not correlate with the clinical pregnancy rates (Spearman r = 0.14, P = 0.16, 95% CI ?0.06 to 0.34). No correlation was recorded between HBA and the standard semen parameters. The study showed that HBA is significantly associated with fertilisation in conventional ICSI. The HBA scores were also significantly associated with the fertilisation rates and biochemical pregnancies.     

Influence of extended incubation time on Human sperm chromatin condensation,sperm DNA strand breaks and their effect on fertilisation rate

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double‐strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double‐strand breaks (r = ?0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.     

Correlation analysis of the progesterone‐induced sperm acrosome reaction rate and the fertilisation rate in vitro

In this study, we aimed to investigate whether progesterone‐induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty‐six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone‐induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = ?0.053, P > 0.01) or rate of high‐quality embryo development (r = ?0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = ?0.406, P > 0.01), fertilisation rate (r = ?0.148, P > 0.01) or progesterone‐induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone‐induced AR may not be used to indicate fertilisation rate.     

How accurate is sperm morphology as an indicator of sperm function?

Sperm morphology has been consistently correlated with fertilisation success or failure. The clinical relevance of the percentage normal spermatozoa has been a widely discussed topic amongst infertility specialists and scientists. This study aimed to evaluate the role of sperm morphology as an indicator of additional sperm functions among 114 andrology referrals. The sperm functions that were investigated included chromatin packaging quality (CMA3 test (n = 109), zona‐induced acrosome reaction (ZIAR test; n = 36), hemizona assay (HZI; n = 36) and progressive motility (n = 47). Chromatin packaging quality had a negative and significant (P = 0.0001, r = ?0.74) correlation with the percentage normal spermatozoa, while progressive motility had a significant and positive correlation (P = 0.0001, 0.59). Accurate sperm morphology scoring as described by the WHO 2010 manual can therefore be used as an indicator of specific sperm functions.     

Is there any correlation between sperm parameters and chromatin quality with embryo morphokinetics in patients with male infertility?

The main goal was to evaluate the correlation between sperm parameters and chromatin quality with embryo kinetics via time‐lapse monitoring system (TLM). A total of 40 couples involved in the ICSI program as a result of male infertility. For assessment of sperm chromatin and DNA quality, we used aniline blue, toluidine blue, chromomycin A3, acridine orange and terminal transferase‐mediated deoxyuridine triphosphate biotin end labelling assays. All mature oocytes were injected, and the generated zygotes (2PNs) were cultured in TLM. In day 3 after injection, single embryo transfer (SET) was carried out according to the morphology and morphokinetics. The patients were followed up until delivery. There were positive significant correlations between sperm count with CC2 (r = .330, p = .049), T4 (r = .329, p = .038), T6 (r = .342, p = .035) and T7 (r = .374, p = .025). Also, there were positive significant correlations between nonprogressive motility and T2 (r = 0.323, p = .042), T3 (r = .411, p = .013) and T4 (r = .418, p = .007). Regarding the sperm chromatin quality assays, there were negative significant correlations between CMA3 and CC2 (r = ?.272, p = .049) and between acridine orange and T5 (r = ?.221, p = .040). It seems that the abnormal sperm parameters and chromatin alteration affect the normal embryo kinetics in ICSI program.     

An additional marker for sperm DNA quality evaluation in spermatozoa of male partners of couples undergoing assisted reproduction technique (IVF/ICSI): Protamine ratio

The aim of this study was to evaluate the relationship between the protamine ratio (P1/P2), DNA fragmentation of spermatozoa and protamine deficiency. Patients were grouped into fertile (G1; n = 151) and sub‐fertile (G2; n = 121). DNA fragmentation in spermatozoa was analysed by a TUNEL assay (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labelling), and the protamination was determined by CMA3 staining, while Western blot was used to measure protamine P1 and P2. While sperm DNA fragmentation (SDF) and protamine ratio were significantly elevated in G2 compared with G1 (12.31 ± 7.01% vs. 17.5 ± 9.5%; p = .001) and (0.91 ± 0.43 vs. 0.75 ± 0.42; p = .003); respectively, the CMA3 positive showed no difference at all between G1 and G2. In G1, the CMA3 positive correlated negatively with the P1/P2 ratio and SDF (r = ?.586, r = ?.297; p = .001 respectively). In contrast, the protamine ratio correlated positively with SDF (r = .356; p = .001). In G2, no correlation was observed between CMA3 positive, SDF and the P1/P2 ratio but the P1/P2 ratio showed a positive correlation with SDF (r = .479; p = .001). In conclusion, the spermatozoa DNA deterioration was closely associated with abnormal protamination but showed an association with the protamine ratio, more than with CMA3 positive. Therefore, for the evaluation of DNA damage in spermatozoa, the P1/P2 ratio might act as an additional biomarker.     

Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests,and assisted reproduction techniques

The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)‐EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS‐EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy‐positive group compared to the negative group, both in IUI (P = 0.042) and ICSI groups (P = 0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P = 0.015, r = 0.214 and P = 0.014, r = 0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.     

Freeze‐dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays

During the freeze‐drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well‐established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff‐Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze‐dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze‐dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = ?0.134 and r = ?0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.     

Total globozoospermia associated with increased frequency of immature spermatozoa with chromatin defects and aneuploidy: a case report

Globozoospermia, characterised by the presence of round spermatozoa lacking acrosomes in an ejaculate, is a known cause of male infertility. Semen analysis, including sperm chromatin structure assay, toluidine blue, chromomycin A3 and aniline blue staining and fluorescence in situ hybridisation, was performed in an infertile globozoospermic patient to establish to which extent these genetic factors contributed to his infertility. No spermatozoa capable of hyaluronan (HA) binding were detected in the HA binding assay. Increased rates of immature spermatozoa with defective replacement of histones by protamines, DNA breaks and disturbed chromatin integrity and sperm aneuploid for the sex chromosomes were observed. Intracytoplasmic sperm injection (ICSI) was used in three in vitro fertilisation (IVF) cycles, and enough morphologically well‐developing embryos were obtained in each cycle. However, no pregnancy was achieved. The infertility of our couple, resistant to IVF/ICSI treatment, was most probably caused by a combination of male and female factors.     

Frequency of sex chromosomal disomy in spermatozoa of normal and oligozoospermic Iranian patients and its effects on fertilisation and implantation rates after ICSI

Chromosomal aneuploidy is a well‐known phenomenon in human gametes including spermatozoa. Success rate of fertilisation and implantation in subfertile patients with male factor has always been shown to be very low. We tried to relate the possible impact of sex chromosomal aneuploidy in spermatozoa used for intracytoplasmic sperm injection (ICSI) on fertilisation and implantation rate. To evaluate the frequency of disomy for X and Y chromosomes in sperm samples retrieved from normal and oligozoospermic individuals, primed in situ labelling (PRINS) technique was used. Following ICSI, the rate of eight‐cell embryos for each category was determined and followed up for successful implantation. Results showed a statistically significant higher frequency of disomy for all chromosomes under study in spermatozoa of oligozoospermic patients compared with normal men (P < 0.01). The rate of eight‐cells embryo formation was significantly lower than in normal group (P < 0.01). The number of embryos transferred for both groups were nearly similar. Implantation rate for oligozoospermic patients was much lower than that of the normal group but was not significantly different (P > 0.05). These results demonstrate that men especially with severe oligozoospermia have an elevated risk for chromosome abnormalities in their spermatozoa. These abnormalities might affect fertilisation and pre‐embryo formation with less impact on implantation.     

Testicular spermatozoon is superior to ejaculated spermatozoon for intracytoplasmic sperm injection to achieve pregnancy in infertile males with high sperm DNA damage

The purpose of this study was to compare the clinical outcome of testicular spermatozoon versus ejaculated spermatozoon in the treatment of infertile males with high sperm DNA damage, referred as sperm DNA fragmentation index (DFI), that attending intracytoplasmic sperm injection (ICSI) programme in terms of clinical pregnancy, births delivered as the primary and pregnancy loss and embryo fertilisation as the secondary outcome. A total of 102 males fulfilling the inclusion criteria were enrolled in the present study. Of the 102 males, 61 infertile males underwent testicular spermatozoon combined with ICSI while the remaining 41 males applied ejaculated spermatozoa in their first ICSI cycles, and the data of them were collected and analysed. In a 18‐month follow‐up, testicular spermatozoon achieved higher pregnancy rate and deliver rate than those in ejaculated sperm group (pregnancy rate, 36% vs. 14.6%, p = 0.017; deliver rate, 38.5% vs. 9.8%, p = 0.001). Nevertheless, there were no significant differences in the number of oocytes aspirated and number of embryos transferred between the two groups. Additionally, the fertilisation rate in the testicular sperm study cohort (70.4%) was also similar to that in the ejaculated sperm group (75.0%). Based on the current data, we conclude that testicular spermatozoon is the prior option in the treatment of infertile males with high sperm DFI in ICSI programme. More high‐quality studies with larger samples size are needed in the future due to the relative small size and the nonrandomized design of the present study.     

N‐acetyl‐L‐cysteine pre‐treatment protects cryopreserved bovine spermatozoa from reactive oxygen species without compromising the in vitro developmental potential of intracytoplasmic sperm injection embryos

Excess of reactive oxygen species (ROS) on in vitro embryo production systems negatively affects the quality and developmental potential of embryos, as result of a decreased sperm quality and increased DNA fragmentation. This issue is of major importance in assisted fertilisation procedures such as intracytoplasmic sperm injection (ICSI), because this technique does not allow the natural selection of competent spermatozoa, and therefore, DNA‐damaged spermatozoa might be used to fertilise an egg. The aim of this study was to investigate a new strategy to prevent the potential deleterious effect of ROS on cryopreserved bovine spermatozoa. We evaluated the effect of a sperm pre‐treatment with different concentrations of N‐acetyl‐L‐cysteine (NAC) on ROS production, viability and DNA fragmentation and assessed the effect of this treatment on the in vitro developmental potential and quality of embryos generated by ICSI. The results show a strong scavenging effect of 1 and 10 mm NAC after exposure of spermatozoa to a ROS inducer, without compromising the viability and DNA integrity. Importantly, in vitro developmental potential and quality of embryos generated by ICSI with spermatozoa treated with NAC were not affected, confirming the feasibility of using this treatment before an ICSI cycle.     

Nuclear sperm quality in total polymorphic teratozoospermia and its impact on intracytoplasmic sperm injection outcome

Various nuclear sperm alterations are reported in patients with syndromic teratozoospermia; however, this has not been clearly identified yet in total polymorphic teratozoospermia. The aim of this study was to analyse sperm aneuploidy, DNA integrity and chromatin packaging in 45 infertile patients with total polymorphic teratozoospermia, and to compare obtained results with those collected from 25 fertile men. For 14 patients, the impact of nuclear sperm abnormalities on intracytoplasmic sperm injection (ICSI) outcomes was analysed. Sperm chromatin condensation was evaluated using aniline blue staining, DNA fragmentation by TUNEL assay and chromosome abnormalities by FISH. The mean DNA fragmentation index was significantly higher in patients compared to controls, weakly and positively correlated to acrosome defects (r = 0.3; p = 0.04) and positively and moderately correlated to microcephalic heads (r = 0.5; p = 0.027). The aniline blue‐reacted spermatozoa rate was also high in comparison with controls, moderately and negatively correlated to progressive motility (r = ?0.6; p = 0.014). Total aneuploidy rate was considerably higher in our patients. A positive and moderate correlation was found between disomy Y rate and acrosome abnormalities (r = 0.5; p = 0.048). These patients had an impaired sperm nuclear quality, which will affect the results in ICSI. Therefore, analysis of sperm chromatin condensation, DNA integrity and aneuploidy in such cases is very useful before ART.     

Combination of running exercise and high dose of anabolic androgenic steroid,nandrolone decanoate,increases protamine deficiency and DNA damage in rat spermatozoa

High doses of anabolic‐androgenic steroids (AAS) are used by some athletes to increase muscle mass, that is often associated with male infertility. The aim of this study was to investigate the possible cause/s of male infertility using a rat model by analysing sperm quality, including its protamine content and DNA integrity, as well as pregnancy rate. Five groups of male Wistar rats were treated for 10 weeks as follows: nandrolone decanoate (10 mg kg^?1 per week) (ND); running exercise (50 min per day, 5 days a week) (EX); Combination of ND and exercise (ND‐EX); nandrolone decanoate solvent (Sham); and control without any injection or exercise (CO). Deterioration in sperm quantity was observed in all test groups (P ≤ 0.01). The frequency of fertile rats was decreased in the ND‐EX and ND groups (P ≤ 0.05). Chromomycin‐A3 staining showed a protamine deficiency in the epididymal spermatozoa in the ND‐EX rats (P ≤ 0.05). Chromatin analysis indicated an abnormal maturation of the sperm nuclei in all test groups compared with the controls (P ≤ 0.05). TUNEL analyses showed a highly significant increase in apoptosis in the EX, ND, and ND‐EX groups (P ≤ 0.01). Our data show that a combination of exercise and high doses of nandrolone decanoate negatively influences the DNA integrity and protamine content resulting in lower sperm quality and reduced pregnancy rate.     

Insight into curcumin nanomicelle‐induced derangements in male reproduction potential: An experimental study

This study was conducted to investigate the possible effects of nanomicelle curcumin (NMC) on spermatogenesis, sperm parameters and in vitro fertilisation potential. For this purpose, 24 mature male Wistar rats were divided into control and test groups. The animals in test groups received 7.5, 15 and 30 mg kg b.w^?1 of NMC (NO = 6 rats in each group). Following 48 days, the DNA integrity of testicular tissues, tubular differentiation (TDI) and spermiogenesis (SPI) indices, sperm parameters and DNA integrity were analysed. Finally, the in vitro fertilisation potential was investigated via evaluating pre‐implantation embryo generation. The NMC diminished the TDI and SPI ratios. The animals in NMC‐received groups exhibited a remarkable (p < .05) reduction in percentage of alive and motile spermatozoa. Moreover, the NMC enhanced the percentage of spermatozoa with decondensed chromatin and elevated the sperm DNA damage ratio. The testicles of NMC‐received groups exhibited severe DNA fragmentation. The percentages of zygote, 2‐cell, blastocysts and hatched embryos generation were decreased in NMC‐received groups compared to control animals. In conclusion, the NMC adversely affects the spermatogenesis and spermiogenesis processes, which in turn results in reducing the sperm quality. Ultimately, decreased sperm quality results in lower pre‐implantation embryo development.     

DNA hydroxymethylation rate in the AChE and HoxC4 promoter associated with human sperm quality

The relationship of altered DNA 5′‐hydroxymethylation in human spermatozoa with seminal parameters remains unclear. The aim of the study was to investigate the association between the 5′‐hydroxymethylcytosine (5hmC) rate in the promoters of acetylcholinesterase (AChE) and homeobox C4 (HoxC4) genes and human sperm concentration/motility. The study population consisted of three groups: asthenozoospermia (AZ), oligoasthenozoospermia (OAZ) and normozoospermia (NZ). The 5hmC rate in the promoter was measured by CCGG loci‐dependent MspI/HpaII restriction mapping of glycosylation‐modified sperm DNA combined with a hydroxymethylation‐specific real‐time polymerase chain reaction assay. The 5hmC rate in the AChE promoter in group AZ and OAZ was higher than that in group NZ (p < .05). A weak inverse correlation between 5hmC rate of AChE and sperm motility was observed in all subjects (r = ?.172, p < .05). The 5hmC rate in the HoxC4 promoter in group OAZ was lower than that in group NZ (p < .05). These results indicated that altered 5hmC rates of AChE and HoxC4 promoters are associated with low sperm motility and sperm concentration respectively.     

Development a nomogram to predict fertilisation rate of infertile males with borderline semen by using semen parameters combined with AMH and INHB

The sperm quality of some males is in a critical state, making it hard for clinicians to choose the suitable fertilisation methods. This study aimed to develop an intelligent nomogram for predicting fertilisation rate of infertile males with borderline semen. 160 males underwent in vitro fertilisation (IVF), 58 of whom received rescue ICSI (R-ICSI) due to fertilisation failure (fertilisation rate of IVF ≤30%). A least absolute shrinkage and selection operator (LASSO) regression analysis identified sperm concentration, progressively motile spermatozoa (PMS), seminal plasma anti-Müllerian hormone (spAMH), seminal plasma inhibin (spINHB), serum AMH (serAMH) and serum INHB (serINHB) as significant predictors. The nomogram was plotted by multivariable logistic regression. This nomogram-illustrated model showed good discrimination, calibration and clinical value. The area under the receiver operating characteristic curve (AUC) of the nomogram was 0.762 (p < .001). Calibration curve and Hosmer–Lemeshow test (p = .5261) showed good consistency between the predictions of the nomogram and the actual observations, and decision curve analysis showed that the nomogram was clinically useful. This nomogram may be useful in predicting fertilisation rate, mainly focused on new biomarkers, INHB and AMH. It could assist clinicians and laboratory technicians select appropriate fertilisation methods (IVF or ICSI) for male patients with borderline semen.     

Different outcomes after intracytoplasmic sperm injection without oocyte activation in two patients with different types of globozoospermia

Different outcomes after intracytoplasmic sperm injection (ICSI) without oocyte activation in two patients with different types of round‐headed spermatozoa (globozoospermia) are reported. After controlled ovarian hyperstimulation and oocyte pick‐up, retrieved oocytes were underwent ICSI without oocyte activation and a 33.33% (4/12) fertilisation rate was obtained in the first case, whereas an abnormal fertilisation was achieved in the second case. The transfer of two grade II embryos in the first couple resulted in clinical pregnancy with a healthy livebirth. It was concluded that the main problem of cases with globozoospermia was a low fertilisation rate or failure fertilisation, and even though ICSI and artificial oocyte activation have been employed to increase this rate, it is not necessarily needed to achieve a pregnancy.