Intracytoplasmic sperm injection (ICSI) has become the most commonly used method of fertilization in assisted reproductive technology. The primary reasons for its popularity stem from its effectiveness, the standardization of the procedure, which means that it can easily be incorporated into the routine practice of fertility centres worldwide, and the fact that it can be used to treat virtually all forms of infertility. ICSI is the clear method of choice for overcoming untreatable severe male factor infertility, but its (over)use in other male and non-male factor infertility scenarios is not evidence-based. Despite all efforts to increase ICSI efficacy and safety through the application of advanced sperm retrieval and cryopreservation techniques, as well as methods for selecting sperm with better chromatin integrity, the overall pregnancy rates from infertile men remain suboptimal. Treating the underlying male infertility factor before ICSI seems to be a promising way to improve ICSI outcomes, but data remain limited. Information regarding the health of ICSI offspring has accumulated over the past 25 years, and there are reasons for concern as risks of congenital malformations, epigenetic disorders, chromosomal abnormalities, subfertility, cancer, delayed psychological and neurological development, and impaired cardiometabolic profile have been observed to be greater in infants born as a result of ICSI than in naturally conceived children. However, as subfertility probably influences the risk estimates, it remains to be determined to what extent the observed adverse outcomes are related to parental factors or associated with ICSI.

Intracytoplasmic sperm injection for male infertility and consequences for offspring

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying > 13.0% total head area, isolated under high magnification (× 6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles

BACKGROUND An embryo's ability to grow and implant can be improved by selection of a normal spermatozoon with a vacuole-free head. However, large vacuoles in spermatozoa have yet to be fully characterized. The present study aimed to determine whether these vacuoles are of nuclear, membrane and/or acrosomal origin. METHODS We studied 15 infertile patients with differing sperm profiles. For each sperm sample, we used high-magnification (×10 000) contrast microscopy to select and assess 30 normal 'top' spermatozoa and 30 spermatozoa with a large sperm-head vacuole (≥ 25% of the head's cross-sectional area). We subsequently analysed the spermatozoa's degree of chromatin condensation (aniline blue staining), DNA fragmentation (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay) and chromosome content (fluorescence in situ hybridization X,Y,18). Atomic force microscopy enabled us to map the plasma sperm membrane in detail. Three-dimensional deconvolution microscopy enabled us to reconstruct images of the nucleus and acrosome in 'top' and 'vacuolated' spermatozoa. RESULTS We studied a total of 450 'top' spermatozoa and 450 vacuolated spermatozoa. The rate of non-condensed chromatin was higher for 'vacuolated' spermatozoa than for 'top' spermatozoa (36.2 ± 1.9 versus 7.6 ± 1.3%, respectively; P < 0.0001). 'Top' and 'vacuolated' spermatozoa did not differ significantly in terms of DNA fragmentation (0.7 ± 0.4 versus 1.3 ± 0.4% respectively; P = 0.25) or aneuploidy (1.1 ± 0.5 versus 2.2 ± 0.7% respectively; P = 0.21). The majority of aneuploid spermatozoa (9 out of 15) lacked chromatin condensation. In all vacuolated spermatozoa, the acrosome was intact, the plasma membrane was sunken but intact and the large vacuole was identified as an abnormal, 'thumbprint'-like nuclear concavity covered by acrosomal and plasmic membranes. CONCLUSIONS The large vacuole appears to be a nuclear 'thumbprint' linked to failure of chromatin condensation.

Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation

Summary


The aim of this investigation was to determine the presence of abnormal sperm chromatin packaging in spermatozoa with large nuclear vacuoles (LNV) selected via high magnification by analysing the pattern of chromomycin A3 (CMA3) staining. A prospective observational study was designed to analyse semen samples obtained from 66 men undergoing infertility diagnosis and treatment. The numbers of cells with normal (dull yellow staining of the sperm head/CMA3-negative) and abnormal (bright yellow fluorescence of the sperm head/CMA3-positive) chromatin packaging were determined on slides with normal and LNV spermatozoa. The presence of bright yellow fluorescence (CMA3-positive) was significantly higher (p < 0.0001) in spermatozoa with LNV than in normal spermatozoa (719/1351; 53.2% vs. 337/835; 40.3%, respectively), reflecting a higher percentage of abnormal chromatin packaging in spermatozoa with large LNV. Our data support the hypothesis that the presence of LNV reflects the presence of abnormal chromatin packaging, which may facilitate sperm DNA damage. As sperm nuclear vacuoles are evaluated more precisely at high magnifications using motile sperm organelle morphology examination (MSOME), the present results support the use of high-magnification sperm selection for intracytoplasmic sperm injection (ICSI).

/pdf/large-nuclear-vacuoles-are-indicative-of-abnormal-chromatin-2rgra0bva2.pdf

Large nuclear vacuoles are indicative of abnormal chromatin packaging in human spermatozoa

Embryo quality is strongly dependent on the in-vitro culture environment. Conventionally, IVF/intracytoplasmic sperm injection (ICSI) embryos are examined microscopically every morning (from day 1 to day 6) to assess fertilization, cleavage and embryo quality. Consequently, the frequent exposure to non-optimal conditions outside the incubator may adversely affect embryonic viability and quality. Hence, this study investigated whether reduction of observation frequency outside the incubator can enhance blastocyst formation rate. A total of 285 IVF/ICSI cycles were divided into two groups. Embryos in the control group (103 cycles) were assessed out-of-incubator every day after insemination (day 1 to day 6; six times). In the experimental group (182 cycles), embryos were assessed four times, on days 1, 3, 5 and 6. The total blastocyst formation rate, day-5 blastocyst formation rate, proportion of good blastocysts and number of cryopreserved blastocysts per patient were significantly lower for the control group compared with the experimental group (42.5%, 31.4%, 50.7%, 1.72±1.55 versus 52.6%, 40.7%, 60.1%, 2.64±2.59, respectively, P

https://www.rbmojournal.com/article/S1472-6483(09)00340-X/pdf

Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate

Spermatozoa selection at high magnification before intracytoplasmic sperm injection seems to be positively associated with pregnancy rates after day 3 embryo transfers. The aim was to demonstrate an association between the presence of vacuoles in sperm nuclei and the competence of embryos to develop to day 5. Grading of spermatozoa at x 6000-x 12,500 magnification: grade I, no vacuoles; grade II, or=1 large vacuole; grade IV, large vacuoles with other abnormalities. The outcome of embryo development in a group of 25 patients after sibling oocyte injection with the four different grades of spermatozoa showed no significant difference in embryo quality up to day 3. However, the occurrence of blastocyst formation was 56.3 and 61.4% with grade I and II spermatozoa respectively, compared with 5.1% with grade III and 0% with grade IV respectively (P < 0.001). Spermatozoa selection at high magnification using Nomarski interference contrast is useful to identify more precisely the size and the number of nuclear vacuoles that greatly exert a negative effect on embryo development to the blastocyst stage. These observations confirm previous studies pointing to possible 'early and late paternal effects', both of which may have an impact on early embryonic development.

https://www.rbmojournal.com/article/S1472-6483(10)60308-2/pdf

Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.

Two-dimensional transvaginal ultrasound (2D) is typically performed to monitor follicle growth in IVF and to determine the optimal time for administering human chorionic gonadotrophin. However, 2D only provides an approximation of the real volume of follicles and therefore cannot be used to guarantee standards for follicular measurement. The automated measurement of follicular size in three dimensions (3D) using a software programme that identifies and quantifies hypoechoic regions within a 3D dataset might provide an objective, fast, valid and reliable standard for such measurements. A prospective controlled study (group I: 20 patients, 2D; group II: 20 patients, 3D) investigated how the criteria for triggering oocyte maturation that are normally used in 2D compare to the new and more accurate method of measuring follicles using 3D-based automated volume count. Significantly more oocytes were fertilized (group 1: 7.1 ± 4.5, group 2: 11.5 ± 6.4; P < 0.03) when using 3D technology and automated volume count. The study assumes that the automated volume count more closely mirrors the biological reality, which means that it can also be used to guarantee the quality standards established by the European Union directive on tissues and cells (2004/23/EC). This new technology therefore holds great promise of becoming the new standard for monitoring follicular growth in IVF.

https://www.rbmojournal.com/article/S1472-6483(09)00072-8/pdf

Improved monitoring of ovarian stimulation using 3D transvaginal ultrasound plus automated volume count

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Blastocyst development after sperm selection at high magnification is associated with size and number of nuclear vacuoles.

Improved monitoring of ovarian stimulation using 3D transvaginal ultrasound plus automated volume count