Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r² = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r² = 0.62, P < 0.05) but less with overall IVF rates (r² = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r² = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.     

MALDI-TOF mass spectrometry rapid pathogen identification and outcomes of patients with bloodstream infection: A systematic review and meta-analysis

There was inconsistent evidence regarding the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for microorganism identification with/without antibiotic stewardship team (AST) and the clinical outcome of patients with bloodstream infections (BSI). In a systematic review and meta-analysis, we evaluated the effectiveness of rapid microbial identification by MALDI-TOF MS with and without AST on clinical outcomes. We searched PubMed and EMBASE databases from inception to 1 February 2022 to identify pre–post and parallel comparative studies that evaluated the use of MALDI-TOF MS for microorganism identification. Pooled effect estimates were derived using the random-effects model. Twenty-one studies with 14,515 patients were meta-analysed. Compared with conventional phenotypic methods, MALDI-TOF MS was associated with a 23% reduction in mortality (RR = 0.77; 95% CI: 0.66; 0.90; I² = 35.9%; 13 studies); 5.07-h reduction in time to effective antibiotic therapy (95% CI: −5.83; −4.31; I² = 95.7%); 22.86-h reduction in time to identify microorganisms (95% CI: −23.99; −21.74; I² = 91.6%); 0.73-day reduction in hospital stay (95% CI: −1.30; −0.16; I² = 53.1%); and US$4140 saving in direct hospitalization cost (95% CI: $-8166.75; $-113.60; I² = 66.1%). No significant heterogeneity sources were found, and no statistical evidence for publication bias was found. Rapid pathogen identification by MALDI-TOF MS with or without AST was associated with reduced mortality and improved outcomes of BSI, and may be cost-effective among patients with BSI.     

Sperm DNA damage and its role in IVF and ICSI

While the semen analysis has traditionally been relied upon to differentiate fertile and infertile men, its utility has been questioned in the current era of assisted reproductive technologies. The desire for more sophisticated diagnostic and predictive tools has led to increased use of sperm DNA damage in the management of male infertility. Despite the availability of numerous assays to measure sperm DNA damage, our understanding of the etiology, measurement, and clinical implications of sperm DNA damage remains incomplete. While the current evidence is fraught with heterogeneity that complicates attempts at comparison and meta-analysis, there does appear to be a role for sperm DNA damage in the development and maintenance of pregnancy in the era of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, as noted by the American Society for Reproductive Medicine, the routine and widespread use of sperm DNA damage testing is not yet supported. Further studies are needed to standardize the measurement of sperm DNA damage and to clarify the exact role of sperm DNA damage within the myriad of other male and female factors contributing to reproductive outcomes in IVF and ICSI.     

Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.     

Relationship between pregnancy,embryo development,and sperm deoxyribonucleic acid fragmentation dynamics

The way the dynamics of DNA fragmentation affects the growth of embryos in real time, and effectiveness of infertility treatment using the ICSI procedure were determined in 148 couples treated with the ICSI technique. The percentage of sperm with fragmented DNA (known as the DNA fragmentation index [DFI]) in semen samples was determined at 3, 6 and 12 h. Embryo culture was assessed continuously during 12 h of observation monitoring.Statistically significant difference was found in DFI at 12 h and outcome of treatment. For the remaining time intervals, no statistically significant differences were noted. An analysis of relationship between the DFI dynamics over time at individual measurements and achievement of pregnancy, confirmed a statistically significant relationship between the rate measured at 6–12 h of observations of DFI changes (DFI 12 h%/h), and achieving pregnancy. Correlation was observed between DFI (during 0, 3, 6 and 12 h), the growth rate in DFI, and time of embryo development. A statistically significant relationship was found between the rate from the start to the end of observations of the DFI, and outcome of treatment.Intensity level regarding fragmentation of sperm DNA and its growth rate affected the time of embryo development in the ICSI procedure. The most significant prognostic factor for achieving pregnancy was intensification of sperm DNA fragmentation after 12 h.     

Flow cytometric sorting of human sperm: MicroSort clinical trial update

This report provides a summary of MicroSort^® efficacy in separation of X- from Y-chromosome bearing human sperm (XSort^® and YSort^®, respectively), clinical outcomes, and the sex of the resultant babies when sorted sperm were used for intrauterine insemination (IUI), in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Clinical trial participants were married couples seeking reduced X-linked genetic disorder risk or family balancing. Sperm were stained with Hoechst 33342, sorted by flow cytometry, then used or cryopreserved for subsequent use. Fluorescence in situ hybridization (FISH) analysis determined the post-sort enrichment (purity) for X- and Y-bearing sperm. Birth and pediatric records were evaluated for incidence of congenital malformations. Between June 1994 and January 2007, patients underwent 3629 IUI cycles, 1642 IVF/ICSI cycles with fresh embryo transfer (ET) and 99 frozen embryo transfer (FET) cycles after MicroSort^®. Of 5871 total sorts, 74.9% were XSort^® and 25.1% were YSort^®. IVF/ICSI fertilization rate was 70.7% and 93.8% of 2PN embryos cleaved. The pregnancy rates for IUI, IVF/ICSI, and FET were 15.6, 32.0, and 33.3%, respectively, while miscarriage rates were 15.7, 14.3, and 33.3%, respectively. Post-sort purity averaged 87.9% (XSort^®) and 73.4% (YSort^®). A total of 1125 clinical pregnancies yielded 943 babies born and 167 ongoing pregnancies. For babies born, XSort^® resulted in 92.0% females and YSort^® yielded 81.5% males. Postnatal follow-up showed a 2.6% major congenital malformation rate, with no recurrent pattern or clustering of malformations. FISH results confirmed MicroSort^® enrichment of X- and Y-bearing sperm populations that closely corresponded with the sex of the resultant child. Fertilization, cleavage, spontaneous abortion, and pregnancy rates as well as incidence of major congenital malformations were comparable to those in literature reports utilizing unsorted sperm.     

Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity

The freeze–thaw process results in reduced motility, viability and fertilization potential of human spermatozoa. So, a variety of substances were evaluated in order to enhance human sperm resistance to the stress of cryopreservation, such as Pentoxifylline (PTX) for improving the Intracytoplasmic sperm injection (ICSI) outcomes. The aim was to investigate the effect of PTX on sperm parameters and chromatin/DNA integrity of asthenozoospermic semen post vitrification. A total of 30 semen specimens were obtained from infertile men with asthenozoospermia. The cryoprotectant-free vitrification was performed for the samples after assessment of sperm parameters. After warming, each sample was exposed for 30 min to 3.6 mmol/l PTX in experimental group and the control group without any treatment apposing at 37 °C for 30 min in regard, to repeat all in vitro analysis (sperm parameters and DNA integrity assay). Regardless of the vitrification devastating impacts on sperm parameters, incubation of post vitrified samples with PTX increased the rate of progressive motility (P < 0.01). Moreover, PTX addition did not significantly damage DNA integrity of asthenozoospermic sperm samples. The data showed that PTX was able to improve sperm movement without any adverse effects on sperm chromatin/DNA integrity in vitrification program.     

Deficiency in mouse Y chromosome long arm gene complement is associated with sperm DNA damage

Background  

Mice with severe non-PAR Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injection (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. In the present study we tested if NPYq deficiency is associated with sperm DNA damage - a known cause of poor ICSI success.     

Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen

An association between sperm DNA integrity and fertility was recently shown for frozen–thawed Norwegian Red (NRF) bull semen diluted in skimmed milk egg yolk (SMEY). In general the fertility of NRF cattle is high, however, in comparison with NRF semen in SMEY, NRF semen diluted in Tris EY based extenders has shown reduced fertility. The aim of the present study was to do a split-sample comparison of sperm DNA integrity of NRF bull semen (n = 20) in SMEY and Triladyl^® (Tris EY based) during routine cryopreservation procedure and during in vitro incubation of frozen–thawed semen in modified synthetic oviduct fluid (mSOF). In contrast to the high fertility of NRF cattle, Holstein cattle are experiencing a marked decline in fertility. Therefore, the present study also aimed to compare sperm DNA integrity of NRF (n = 20) and Holstein (n = 20) semen diluted in Triladyl^® during in vitro incubation. The sperm DNA integrity was measured by susceptibility to in situ acid induced denaturation by the Sperm chromatin structure assay (SCSA). Compared to initial values of frozen neat semen, an increase in DNA damage was observed after dilution and cooling (5 °C) and after freezing–thawing of NRF semen in SMEY, but only after freezing–thawing for NRF semen diluted in Triladyl^®. Sperm DNA damage of NRF semen increased during in vitro incubation in mSOF; the increase in percentage of spermatozoa with DNA damage was more prominent in SMEY than in Triladyl^®, while the degree of damage was higher in Triladyl^®, throughout the incubation period. However, while the correlation between DNA damage and sperm survival was negative in SMEY throughout the incubation period, a positive correlation was observed in Triladyl^® after 9 h of incubation, indicating a higher presence of DNA damage in the live sperm population. In comparison with Holstein spermatozoa, the sperm DNA integrity of NRF semen reflected a better ability to withstand alterations induced during in vitro incubation in mSOF. In conclusion, sperm DNA integrity of NRF bull semen was altered during the cryopreservation procedure and in vitro incubation in mSOF. Dilution in Triladyl^® maintained bull sperm DNA integrity better than dilution in SMEY. Furthermore, alterations in Holstein sperm DNA integrity was more pronounced during in vitro incubation in mSOF compared to NRF bull spermatozoa.     

Analysis of birth defects among children 3 years after conception through assisted reproductive technology in China

BACKGROUND : Previous studies inconsistently suggest that assisted reproduction technology (ART) may increase the risk of birth defects in children. METHOD(S) : Live birth infants, conceived by in vitro fertilization fresh embryo transfer (IVF), intracytoplasmic sperm injection fresh embryo transfer (ICSI), or frozen‐thawed embryo transfer (FET) in Reproductive Center of Tongji Hospital (Wuhan, China) between 1997 and 2008, were followed up at birth and after 3 years. Preterm pregnancy, multiple pregnancy, sex ratio (male/female), congenital malformation were compared. RESULT(S) : A total of 4,236 children were born after ART (IVF 2,543, ICSI 908, FET 785). Compared with IVF, the rate of preterm pregnancy and sex ratio in ICSI were lower (p < 0.05); the rate of multiple pregnancy in ICSI and FET were all lower than IVF (p < 0.05). Congenital defects were comparable in all groups at birth. In total, 2,908 children participated in the second follow‐up from 34 months to 60 months with an average of 40 months, and the cases of birth defects had doubled (3 years: 5.16%, birth: 2.22%). The birth defect rate in boys conceived through ICSI was significantly higher than the IVF group after 3‐year follow‐up (ICSI boys: 8.62%, IVF boys: 5.21% [p < 0.05]), even though there was no significant difference at birth. CONCLUSION(S) : Compared with IVF, FET may not increase risk of birth defects. Children conceived through ICSI, especially males, had higher rates of congenital malformations that were inapparent at birth. So longitudinal monitoring may provide insights into the risks of ART. Birth Defects Research (Part A) 97:744–749, 2013. 2013. © 2013 Wiley Periodicals, Inc.     

Early-cleavage is a reliable predictor for embryo implantation in the GnRH agonist protocols but not in the GnRH antagonist protocols

Background  

To test if early-cleavage was a strong predictor of pregnancy in patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol for in-vitro fertilization treatment (IVF) and intracytoplasmic sperm injection (ICSI).     

Blood metal/metalloid concentration of male subjects undergoing IVF/ICSI treatment outcomes: A prospective cohort study

BackgroundPrevious epidemiology studies reported that heavy metal/metalloid exposure is associated with the impairment of semen quality. However, it is still not clear whether the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment outcome will be affected after the heavy metal/metalloid exposure of the male partners.MethodsA prospective cohort study with a 2-year followed-up was conducted in a tertiary IVF center. A total of 111 couples undergoing IVF/ICSI treatment were initially recruited from November 2015 to November 2016. Male blood concentrations of heavy metal/metalloid including Ca, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Mo, Cd, Hg, and Pb were measured by inductively coupled plasma mass spectrometry, and the lab and pregnancy outcome data were followed up. The associations between male blood heavy metal/metalloid concentration and the clinical outcomes were analyzed by Poisson regression analysis.ResultsOur results showed that none of the heavy metal/metalloid of male partners we investigated are significantly associated with the oocyte fertilization and good embryo (P ≥ 0.05); however, antral follicle count (AFC) was a protective factor for the oocyte fertilization (RR: 1.07, 95 % CI: 1.04–1.10). The blood Fe concentration of the male partner was positively associated (P < 0.05) with pregnancy in the first fresh cycle (RR:170.93, 95 % CI: 4.13–7082.04), cumulative pregnancy (RR: 23.61, 95 % CI: 3.25–171.64) and cumulative live birth (RR: 36.42, 95 % CI: 1.21–1092.54). In the first frozen embryo cycles, pregnancy was significantly associated (P < 0.05) with the blood Mn (RR: 0.01, 95 % CI:0.00–0.11) and Se concentration (RR: 0.01, 95 % CI:8.25 E-5–0.47) and female age (RR: 0.86, 95 % CI:0.75–0.99); live birth was significantly associated (P < 0.05) with the blood Mn concentration (RR: 0.00, 95 % CI: 1.14E-7–0.51).ConclusionsOur results suggested that the higher male blood Fe concentration was positively associated with pregnancy in the fresh embryo transfer cycle, cumulative pregnancy, and cumulative live birth, whereas the higher male blood Mn and Se concentration were associated with lower chance of pregnancy and live birth in the frozen embryo transfer cycle. However, the underline mechanism of this finding still needs further investigation.     

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization,perivitelline, and intracytoplasmic sperm injections

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4′,6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.     

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R² value = 0.949; P < 0.01; n = 16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n = 6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 °C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P = 0.001) in DNA damage, as soon as 4 h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6 h of incubation and revealed individual animal variation. Freshly collected and incubated (37 °C) rhinoceros (n = 3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.     

Sperm quality and paternal age: effect on blastocyst formation and pregnancy rates

Background

Several studies suggest a decrease in sperm quality in men in the last decades. Therefore, the aim of this work was to assess the influence of male factors (sperm quality and paternal age) on the outcomes of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Methods

This retrospective study included all couples who underwent IVF or ICSI at Montpellier University Hospital, France, between 1 January 2010 and 31 December 2015. Exclusion criteria were cycles using surgically retrieved sperm or frozen sperm, with pre-implantation genetic diagnosis or using frozen oocytes. The primary outcomes were the blastulation rate (number of blastocysts obtained at day 5 or day 6/number of embryos in prolonged culture at day 3) and the clinical pregnancy rate. The secondary outcomes were the fertilization and early miscarriage rates.

Results

In total, 859 IVF and 1632 ICSI cycles were included in this study. The fertilization rate after ICSI was affected by oligospermia. Moreover, in ICSI, severe oligospermia (lower than 0.2 million/ml) led to a reduction of the blastulation rate. Reduced rapid progressive motility affected particularly IVF, with a decrease of the fertilization rate and number of embryos at day 2 when progressive motility was lower than 32%.Paternal age also had a negative effect. Although it was difficult to eliminate the bias linked to the woman’s age, pregnancy rate was reduced in IVF and ICSI when the father was older than 51 and the mother older than 37 years.

Conclusions

These results allow adjusting our strategies of fertilization technique and embryo transfer. In the case of severe oligospermia, transfer should be carried out at the cleaved embryo stage (day 2–3) due to the very low blastulation rate. When the man is older than 51 years, couples should be aware of the reduced success rate, especially if the woman is older than 37 years. Finally, promising research avenues should be explored, such as the quantification of free sperm DNA, to optimize the selection of male gametes.

     

Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

The objectives of this study were to assess the effects of induced testicular degeneration in Bos taurus indicus (Nellore) bulls on changes in seminal characteristics and fertilizing ability of sperm. Four Nellore bulls (30–36-month-old, 500–550 kg) with good seminal quality (>80% motile and morphologically normal sperm) had scrotal insulation applied for 5 d. Semen was collected by electroejaculation and cryopreserved at the pre-insulation moment, and 7, 14, and 21 d after insulation was removed. Gross motility, vigor of sperm movement (1–5), acrosome integrity, sperm morphology (phase-contrast microscopy), nuclear vacuoles and abnormal chromatin (Feulgen-stain) were determined after sperm preparations for in vitro fertilization (IVF). Prior to IVF, sperm were separated using a Percoll gradient (45% and 90%). Normal sperm decreased (P < 0.05) 14 and 21 d after insulation was removed. On 14 and 21 d, the incidence of head defects (9.7 ± 0.6 and 17.0 ± 0.8, respectively; mean ± S.E.M.) was higher (P < 0.05) in agreement with the incidence of nuclear vacuoles (14.0 ± 5.0 and 12.3 ± 2.3) and abnormal chromatin (24.4 ± 7.2 and 30.8 ± 2.8). Although the frequency of cleaved oocytes decreased only on 21 d (P < 0.05), blastocyst rates were lower (P < 0.05) than pre-insulation on 14 and 21 d. In regression analyses, only nuclear vacuoles, head defects and intact acrosome accounted for differences in cleavage (R² = 0.38, 0.48, and 0.30, respectively) and blastocyst rates (R² = 0.35, 0.37, and 0.44). Abnormal chromatin was associated only with blastocyst rates (R² = 0.35). In conclusion, blastocyst rate was more sensitive than cleavage rate and the assessment of nuclear integrity is recommended to predict the fertilizing ability of bull sperm.     

Iron-induced oxidative DNA damage in rat sperm cells in vivo and in vitro

We investigated whether acute iron intoxication causes oxidative DNA damage, measured in terms of 7-hydro-8-oxo-2′-deoxyguanosine, 8-oxodG, in nuclear DNA in testes and epididymal sperm cells in vivo and in vitro in rats. In addition, we investigated levels of the modified nucleoside in liver and kidney and measured its urinary excretion.

Sperm cells were isolated from the epididymides and the testes cells were isolated after homogenisation. In vitro, the sperm and testes cells were incubated with increasing concentrations of FeCl₂ ranging from 0 to 600 μM. The median (range) levels of 8-oxodG/10⁵ dG in the epididymal sperm cells increased from 0.48 (0.42–0.90) to 15.1 (11.4–17.6) (p < 0.05), whereas the level rose from 0.63 (0.22–0.81) to 8.8 (4.5–11.6) (p < 0.05) at 0 and 600 μM, respectively, in the testicular cells.

In vivo groups of 7–8 rats received 0, 200 or 400 mg iron/kg as dextran i.p. After 24h, epididymal sperm cells, testes, kidneys and liver were collected for analysis. Kidney and sperm DNA showed a significant increase in 8-oxodG in the iron-treated animals. The median (range) values of the 8-oxodG/10⁵ dG in the epididymal sperm cells rose from 0.66 (0.38–1.09) to 1.12 (0.84–5.88) (p < 0.05) at 0 and 400 mg iron/kg, respectively, whereas the values in the testes and liver showed no significant change. In the kidneys the 8-oxodG/10⁵ dG median (range) values were 0.98 (0.73–1.24), 1.21 (1.13–1.69) and 1.34 (1.12–1.66) after 0, 200 and 400 mg iron/kg, respectively (p < 0.05).

The 8-oxodG-excretion rate was measured in 24 h urine before and after iron treatment. The rate of urinary 8-oxodG excretion increased from 129 (104–179) pmol/24 h before treatment to 147 (110–239) pmol/24h after treatment in the group receiving 400 mg iron/kg (p < 0.05).

The results indicate that acute iron intoxication may increase oxidative damage to sperm and kidney DNA.     

Electrochemical detection of natural DNA damage induced by ferritin/ascobic acid/H2O2 system and amplification of DNA damage by endonuclease Fpg

Oppositely charged natural DNA and chitosan (CS) were assembled into (CS/DNA)_n layer-by-layer films on electrode surface, and Ru(bpy)₃²⁺ (bpy = bipyridyl) in solution was used as electroactive catalyst to detect damage of DNA in the films after incubation of the films in ferritin/AA/H₂O₂ solutions (AA = ascorbic acid). The mechanism of DNA damage caused by the ferritin/AA/H₂O₂ system was similar to that of Fenton reaction, where the reaction of ferritin with AA would release some Fe(II) ions from ferritin and the following reaction between Fe(II) ions and H₂O₂ would produce hydroxyl radical, which could induce DNA oxidative damage. This system provided an in vitro model to imitate the DNA damage indirectly induced by ferritin in real bio-systems. In addition, formamidopyrimidine DNA glycosylase (Fpg), a key endonuclease enzyme in repair of oxidatively damaged DNA, was used to amplify the DNA damage caused by ferritin/AA/H₂O₂ system through conversion of oxidative purine bases into single-strand breaks. The high sensitivity of electrocatalytic method with Ru(bpy)₃²⁺ as the catalyst in detection of DNA damage and the magnification function of Fpg may provide a novel idea to detect natural DNA lesion sensitively.     

The treatment of obstructive azoospermia by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) allows the treatment of virtually every type of male infertility. Unlike in vitro fertilization (IVF), its success does not depend on sperm concentration, motility or morphology and most of the physical barriers to fertilisation are by-passes. Since ICSI does not require strongly motile sperm, its use has now been expanded to incorporate immature sperm from the testes and epididymides. Successful fertilisation, pregnancies and healthy babies have all been reported. However, concerns about the safety of ICSI remain due to its short clinical history and the lack of testing on animal models. Male fertility potential for assisted reproduction by ICSI cannot be measured by conventional parameters. Sperm DNA integrity is increasingly recognised as a more useful indicator. Studies have shown that sperm with higher levels of DNA damage have lower fertilisation rates after IVF and ICSI. Sperm with DNA damage above a certain threshold are associated with a longer time to conceive in otherwise apparently fertile couples and a higher miscarriage rate. DNA damage has been shown to be associated with impaired embryo cleavage. Our group has shown that sperm DNA from testicular sperm is less fragmented than that from epididymal sperm and suggest its preferred use in ICSI. In addition to nuclear (n) DNA we also assessed the quality of mitochondrial (mt) DNA from testicular sperm from men with obstructive azoospermia undergoing ICSI. We observed that couples achieving a pregnancy had both less mtDNA deletions and less nDNA fragmentation. We found inverse relationships between pregnancy and sperm mtDNA deletion numbers, size and nDNA fragmentation. No relationships were observed with fertilisation rates. With this knowledge, we designed an algorithm for the prediction of pregnancy based on the quality of sperm nDNA and mtDNA. Each year 40,000 men have a vasectomy in the UK but every year 2500 request a reversal to begin a second family. For such men, vasectomy reversal has recently been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) performed at an outpatient clinic and subsequently used in ICSI. Since these were previously fertile men it has been assumed that they had ‘fertile’ sperm. However the assited conception success rates of these mens partners has not been assessed until recently. We have shown a significant reduction in the clinical pregnancy rates in the partners of men who had had a vasectomy ≥10yrs previously. There is also evidence to suggest that spermatogenesis is significantly impaired in vasectomised men. Marked decreases in spermatocytes, spermatids and spermatozoa have been observed. We have found this to be associated with concomitant increases in apoptotic markers, such as Fas, FasL and Bax. The quality of the remaining sperm is also compromised. Sperm DNA from vasectomized men shows substantial damage which increases with time after surgery. This new use of ICSI will be discussed.     

L’utilisation du MSOME: expérience de six ans

Introduction

MSOME (Motile Sperm Organellar Morphology Examination) is a new method for real-time evaluation of sperm morphology under 6600x high magnification. ICSI modified procedure with sperm selected by MSOME is named IMSI (Intracytoplasmic Morphologically Selected sperm Injection). IMSI has been developed to improve ongoing pregnancy rate in couples with repeated implantation failure.

Material and methods

The study concern an observational cohort of 11535 ICSI performed with fresh ejaculated sperm in our ART lab between January 2004 and July 2009. Among them, 2509 were realized with IMSI. The primary outcome measures were cleavage rate per injected oocyte on day 2, clinical pregnancy and abortion rates. Comparisons were performed using Chi square² test and univariate analysis of variance.

Results

There were no significant difference between conventional ICSI and IMSI groups in term of cleavage and pregnancy rates. Couples with abnormal sperm (teratozoospermia, oligozoospermia and oligoteratozoospermia) and no previous ICSI failure, had a significantly higher clinical pregnancy with IMSI than with ICSI (34.4% vs. 27.1%, p = 0.02). Furthermore, pregnancies obtained in patients with teratozoospermia were associated with a lower abortion rate after IMSI than after ICSI, close to significance (12.6% vs. 19.6%, p = 0.08).

Conclusion

In cases of severe teratozoospermia, IMSI appears to improve pregnancy rate and pregnancy outcome.