Sperm selection for IVF and ICSE based on gross morphology and routine analysis not sensitive

The comet is another technique that qualitatively measures sperm DNA damage by visualising single- and double-strand breaks using electrophoresis. A doublestrand DNA appears at the head of a comet. The full head and tail emerge, while damaged double- and single-strand DNA fragments move towards the tail part. Therefore, this essay is an immensely useful tool to measure the DNA fragmentation index (DFI, %), which indicates the number of cells with DNA damage, and high DNA stainable (HDS) (%), which indicates the proportion of the histone-to-protamine transition in the immature sperm. The disadvantages of this test are that the testing requires expensive equipment and a high concentration of sperm and that the reference range for the sample needs to be calibrated.

Flow cytometry is a potent molecular technique that has the ability in measuring several markers accurately and in a short time. In an andrology laboratory, it can be used to differentiate between several types of DNA abnormalities, apoptosis markers, the detection of antisperm antibodies, and others. FCM principles depend on calculating the ratio of singleand double-stranded DNA staining of sperm nuclei with DNA fluorescent stain. During FCM, exposure to acid usually results in denaturing of double-stranded DNA in spermatozoa whose chromatin structure is altered.

Apoptosis or programmed cell death (PCD) in male germ cells normally occurs in the prospermatogonia (gonocytes) layer in the differentiating testis during fetal life. Apoptosis usually occurs during normal spermatogenesis and accounts to the loss of up to 75% of potential sperm number, where only 25% of the expected number of primary spermatocytes is produced from the spermatogonia A. Impairment of apoptosis at this stage generates a male infertility phenotype, while apoptosis in the mature spermatozoa appears to be significantly involved in the production of a subfertile state

Apoptotic features that detected in the PCD of germ line include the following: condensation of the chromatin, membrane leakage, endoplasmic reticulum Ca2+ pool depletion, release of cytochrome c from mitochondria, downstream caspase activation, generating substrate cleavage, endonuclease activation, and DNA fragmentation.

Semen samples were obtained by masturbation after at least 3 days of abstinence. The samples were subjected to analysis according to the World Health Organization criteria (2010). The study inclusion criteria were applied scheduled for a routine semen analysis as a patient at Princess Al-Jawhara fertility hospital, age 20 years and more, infertility of 1 year or more whether primary or secondary, and not currently receiving hormonal treatment. The study was aimed at assessing the level of DNA fragmentation and apoptosis by measuring flow cytometry using new techniques.

Male partners of 139 infertile couples agreed to participate in the study; only eighty-four of them fulfilled the criteria for inclusion. More than half of the patients (63.1%) were in the age group of 26-35 years. Around 64.3% of patients had normal semen samples.

DNA analysis was not normal in all normozoospermic samples. About 54.8% of the semen samples demonstrated abnormal chromatin status. Samples with normal class one chromatin condensation were represented only 32.1% of the study population. Aneuploidy cells were detected in three samples with an abnormal semen analysis. Sample with abnormal p53 activity had a high level of positively labeled cells with anti-p53 antibodies (≥5%). Also, samples with abnormal Bcl-2 activity had ≥5% of positively labeled cells. Apoptotic markers’ analysis reveals that contrary to expectation, normal chromatin condensation did not reflect standard cell cycle analysis. About 65.5% of the semen samples had an abnormally high level of positively labeled cells with FITC Mouse Anti-Human Bcl-2; some of them had normal chromatin condensation, while 51.2% of the semen samples had an abnormally high level of positively labeled cells with a p53-PE antibody.

Flow cytometry analysis revealed a varying degree of DNA damage. It was able to quantify the degree of impairment even in samples with minimal DNA fragmentation. DNA damage was observed even in samples that were considered normal with a routine semen analysis. Flow cytometry was sensitive to changes in sperm apoptosis. Elevated p53 activity levels were associated with high DNA fragmentation. Meanwhile, B-cell lymphoma 2 (Bcl-2) activities showed a different pattern. The present study had the importance in measuring p53 level in human sperm population by using flow cytometry. It revealed that around 51.2% of the study population had abnormal measures of p53 protein with a significant correlation between the degree of DNA damage and p53 level.

Despite the widespread decline in human fertility, science has not established perfect methods for diagnosis. To date, all the IVF techniques rely in their final sperm selection on its gross morphology, not the nuclear content or DNA normality. Besides, most of the urological procedures rely principally on traditional semen analysis to assess treatment success. The study demonstrated considerable differences in the results of the traditional analysis in comparison to those of the molecular procedures. The cytometry analysis of DNA integrity revealed considerable damage in sperm DNA even within the grossly normal samples. These findings indicate that selection of sperm for IVF and ICSE on the bases of gross morphology and routine analysis is not sensitive. Because of their role in sperm maturity, analysis of apoptotic markers was performed.

“We have many gaps in our knowledge regarding male infertility, partly because of the restricted number of researches in our area in this crucial field and partly because of the limited value of traditional semen analysis in the assessment of infertile males. The study emphasized that future studies should apply the molecular markers in male infertility diagnosis, primarily for those involved in ART cycles. This could have a positive impact on ART results, especially if we correlate the outcome of these procedures with the findings of these assays.”

Source: Huda Mossa Omran, Moiz Bakhiet, and Volker Ehemann; Hindawi International Journal of Reproductive Medicine Volume 2021

https://doi.org/10.1155/2021/9531775