Male factor testing in recurrent pregnancy loss cases: A narrative review

Abstract Recurrent pregnancy loss is a distinct disorder defined as the loss of at least 2 pregnancies before the 20 th wk of gestation. With half of the genome of the embryo belonging to the father, the integrity of the sperm genome is crucial for a successful pregnancy. Semen analysis is recommended for men in such cases to evaluate sperm concentration, morphology, vitality and motility. However, other important sperm parameters such as sperm epigenetics, aneuploidy, Y chromosome microdeletion and chromatin integrity also correlate with successful pregnancy and delivery rate. This article examines the use of different sperm tests and their importance in male partners of women suffering from recurrent pregnancy loss.


Introduction
Men are accountable for 50% of the genomes of an embryo. However, they are usually not considered in clinical studies and the treatment management in recurrent pregnancy loss (RPL) cases (1). For many years, women have been held responsible for all forms of infertility and reproduction defects. Undoubtedly, women play an important role in reproduction and several complications may arise due to female infertility (2). Although many new clinical studies focus on the effect of male health on infertility and abortion (3). There are still not enough studies on the role of paternal factors in RPL (4).
Moreover, very few studies have assessed and demonstrated the role of the male factor and its genetics in cases of recurrent abortion (5). The main diagnostic test to assess male fertility is a simple screening, including standard and routine semen analysis and some genetic testing; however, finding the best tests for diagnosing male infertility remains in debate (6). Paternal age is a known factor in cases of miscarriage, and older age in men can elevate the risk of spontaneous abortion (7). The hypothesis that men are fertile at any age while women's fertility is age-dependent is not totally correct (8,9). Although, the relationship between advanced paternal age and the risk of spontaneous abortion is still unknown and controversial (10,11). Some studies have already shown that the risk of abortion rises with the rise in men's age and decline in male fertility (12,13). However, on the contrary, some studies have shown no increased rate of spontaneous abortion in cases with advanced paternal age (14,15). Other risk factors involved in cases of male infertility are systematic and chronic disease (16), environmental and ecological toxins (17), obesity (18), and lifestyle parameters (19); however, in clinical trials, none of these factors have been thoroughly studied nor has the association between them and increased risk of spontaneous abortion in couples suffering RPL been evaluated.
Therefore, this article focuses on the different sperm tests that are currently available and their importance in determining male fertility in couples suffering RPL.

Materials and Methods
Scientific databases such as PubMed,   (20). However, there is controversy regarding the efficacy of semen analysis in cases of miscarriage (21).

Inclusion and exclusion criteria
In some cases, the abnormal sperm morphology in men has been accepted as a possible etiology of an unsuccessful pregnancy, as fertilization with an abnormal sperm may not always result in a healthy embryo (22,23). In addition, studies of RPL at infertility treatment clinics showed that abortion increased in couples with abnormal hypo-osmotic swelling scores (24,25). It has been suggested that routine sperm analysis is insufficient for detecting sperm abnormality, and other functional individualities of sperms need more examination in RPL cases (26). Interestingly, some of the latest studies on sperm's role in RPL have reported no significant relationship between abnormal sperm parameters including count, morphology and motility, and the risk of RPL (27,28

DNA fragmentation testing
There are several methods for detecting sperm DNA damage (20). However, based on some studies, the role of SDF in evaluating male fertility and in couples who suffer RPL is controversial (32).
Many studies have shown that cases with RPL have a higher percentage of DNA fragmentation index (DFI) in the sperm, and this condition can affect sperm function, embryo formation, and development (1,3,4).
Some studies have shown that in cases with paternal advanced age, there was a higher percentage of DNA fragmentation (33,34), varicocele, toxic exposure, alcohol consumption, and hormonal disorders (20). Moreover, some studies have shown that the level of DFI was higher in infertile men compared to fertile ones; however, documentations for men whose partner had a high risk of abortion were unpredictable (5,35  The TUNEL assay can detect DNA damage (single and double strand) and this test has a high sensitivity and reproducibility when using the flow cytometry method, but it requires fluorescence microscopy or flow cytometry, which are expensive, and it is a prolonged procedure with variable protocols (38)

SCD
The SCD test is a simple and rapid assay. Also, a low number of sperm is needed. However, the assay only detects single-strand breaks with low contrasting images and an inter-observer subjectivity is needed for the interpretation of halos (38).
A study revealed the strongest association between TUNEL and miscarriage among all assays tested. The authors stated that the reason for this difference could be related to the lack of a denaturation step in the TUNEL assay (21). Another study showed that the SCD test in comparison with the TUNEL assay had high sensitivity for the assessment of DNA damage in unexplained infertile men (46). A systematic review demonstrated a significant strong relationship between defects in sperm chromatin quality, especially high levels of sperm DNA damage, and occurrence of RPL using both TUNEL and SCD assays. In addition, when they analyzed the subgroup and assessed the comparison between some sperm fragmentation tests, a similar association was seen with TUNEL and SCD by RPL risk (47     They suggested that protamines may play an additional role in early embryogenesis (70).

Sperm aneuploidy testing
Chromosomal abnormalities in somatic cells can be discovered by a simple blood karyotype analysis. Errors during sperm meiosis lead to chromosomal abnormalities, which cannot be detected by a blood karyotype analysis (71). A common cytogenetic tool for sperm aneuploidy screening is fluorescent in situ hybridization (45

Zidi-Jrah et al. (45)
The sperm progressive motility was significantly lower and abnormal morphology was significantly higher in the RPL group vs. the fertile group

Zhang et al. (50)
There were no significant differences in sperm concentration, motility, and normal morphology between the RSA group and the controls Esquerre-Lamare et al.
Abnormal parameters (morphology and concentration) were found in 25% of the patients

TUNEL assay
Bareh et al. (42) The mean SDF was significantly higher in men with RPL compared with normozoospermic men

Carlini et al. (43)
A correlation between increased SDF and impaired fertilization and pregnancies was noted

Brahem et al. (44)
Men with a history of RPL had a higher incidence of DNA damage and poor motility than men from the control group

Zidi-Jrah et al. (45)
The percentages of SDF and nuclear chromatin decondensation were significantly higher in the RPL group than in fertile men  The level of abnormal DNA fragmentation in the RSA group was significantly higher than in the control group

Ribas-Maynou et al. (49)
The SDF was significantly higher compared to in the controls (p < 0.01)

Zhang et al. (50)
There was no significant difference in sperm chromatin integrity between the RSA group and the controls The percentage of immature spermatozoa from the unexplained RSA group was higher than in fertile men with no history of RSA using AB, TB, AO, and CMA3 staining

Ruixue et al. (58)
The RPL group had a higher mean percentage of AB staining-positive sperm compared with the control group

Nabi et al. (59)
The bacterial contamination was higher in the RPL group than in the fertile group. In addition, AB & TB staining showed that the percentage of abnormal spermatozoa (AB + ) & (TB + ) in the RPL group was higher than in the control group

Nazari et al. (60)
The effect of vitamin E and zinc on sperm chromatin quality was evaluated by AB, TB, and CMA3 staining. It was found that the number of AB, TB, and CMA3-positive sperm decreased significantly after antioxidant therapy

Pourmasumi et al. (4)
The effect of vitamin E and selenium on sperm chromatin quality in couples with RM was evaluated, and it was found that the number of TB, AB, CMA3-positive sperm decreased significantly after antioxidant therapy

Kazerooni et al. (62)
The sperm chromatin quality in couples with SRA was compared to the fertile group. The SRA group had a significantly higher percentage of CMA3 + and AB + sperm than the fertile men, but the AO results in the 2 groups did not differ significantly

Conclusion
Considering the importance of sperm integrity in a successful pregnancy, semen analysis is recommended for men in couples suffering RPL to evaluate sperm concentration, morphology, vitality, and motility. Based on the literature reviewed in this paper, the evaluation of the integrity of the sperm DNA, sperm aneuploidy, and YCM may be useful in guiding the management of RPL cases. Since the relationship between sperm DFI and RPL is being investigated and has been presented in this article, it seems that SDF testing may be a valuable tool for RPL assessment in clinical centers before assisted reproductive techniques.