Sever Oligospermia Treatment with Testicular Sperm Using ICSI

Assisted reproductive technology has been developed significantly throughout the past few years, particularly diagnosing and treating male infertility. Many studies have been performed showing that Intracytoplasmic Sperm Injection (ICSI) is a successful method to attain clinical pregnancy and live birth through impaired spermatozoa characteristics or low sperm count, such as severe oligospermia. Severe oligospermia indicates low sperm count, which in some cases leads to azoospermia. Severe oligospermia can be caused by several factors such as genetics or medication. In search of efficient treatment for couples with Severe oligospermia, numerous retrospective and prospective researches have reported high pregnancy and live birth rates through testicular sperm for men with severe oligospermia and cryptozoospermia with or without high sperm DNA damage. The research showed that the use of testicular sperm in combination with ICSI yielded a high pregnancy rate and live births over another source of sperm, such as ejaculated sperms. Moreover, the use of ICSI in severe oligospermia has shown successful fertilization and pregnancy.


Introduction
Male infertility factors have been recognised in around 40 %-50 % of infertile couples; more than 90% of cases in male infertility are due to low sperm counts, poor sperm quality or both. Other factors involve anatomical disorders, hormonal imbalances and genetic defects [1] (Leaver, 2016). Moreover, male factors can include ejaculatory problems such as retrograde ejaculation, premature ejaculation or anejaculation, defects in sperm count, mobility, or abnormalities [1] (Leaver, 2016). Some of the most common factors that correlate to irregularities in sperm are listed in (Table 1) .
Male infertility has geographical variation in the prevalence of male infertility. High fertility levels can be found in France (59 %), 26 % -32 % in the UK and Kashmir Valley in India, and about 36 % in South Africa, Indonesia and Finland. Moreover, it has been shown that mean sperm concentration has regional variation in men from different regions of the USA and France [2] ( Mehta et al., 2006).
There are different ways of male infertility treatment, depending on the male infertility factors, for instance, improving the quality of sperm, surgery to repair a varicocele or intracytoplasmic sperm injection (ICSI) procedures. ICSI requires injecting one sperm directly into an oocyte prior to transferring to the uterus. ICSI has the advantage of using few viable sperm to fertilise an egg and can bypass natural barriers that prevent fertilisation [1] (Leaver, 2016).
This report will be discussing and evaluating the application of ICSI in treating men with significant oligospermia

Sperm Abnormalities Descriptions
Aspermia Male with no semen

Azoospermia
Male with no sperms in the semen

Low sperm concentration
Oligospermia Low sperm count Teratozoospermia Irregular sperm shape (96% of sperms)

Sever oligospermia
Oligozoospermia is a disorder where sperm concentration is below the lower reference limit of 15 million sperm/ml of ejaculate. Oligozoospermia can be further classified as mild (between 10 and 15 million sperm/ml), moderate oligozoospermia (between 5 and 10 million sperm/ml), and severe oligozoospermia (less than 5 million sperm/ml) [3] (Choy and Amory, 2020). Oligozoospermia diagnosing can be challenging, as several conditions are recognised to result in oligozoospermia. Therefore, a formal and precise diagnosis can be relevant in giving patients a reason for their sterility and decreasing confusion, fear, and stigma [3] (Choy and Amory, 2020).
Disturbance of the regulated hormonal axis can induce the increase to several of the endocrinebased etiologist of oligozoospermia. Usually, the hypothalamus produces and secretes gonadotropin-releasing hormone (GnRH) into the hypothalamic-hypophyseal portal circulation; GnRH influences the anterior pituitary to induce the gonadotroph cells to produce and release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. LH excites the Leydig cells of the testes to deliver testosterone, while FSH stimulates the Sertoli cells to induce spermatogenesis ( Figure 1). Generally, any destruction of gonadotropin secretion will result in a downstream outcome of disrupting intratesticular testosterone biosynthesis and spermatogenesis [3] (Choy and Amory, 2020). Males with infertility have higher chromosomal irregularities than fertile men and usually do not present any phenotypic features. In the oligozoospermic, men of autosomal translocations have a rate of inversions predominate (3%) over sex chromosome anomalies (1.6%). Fertility may be expected, but interference with spermatocyte chromosomal pairing re-results in azoospermia or, more frequently, oligozoospermia (McLachlan, 2013) Yq microdeletions are the most well-known genetic condition of spermatogenic failure.

Genetics factors and oligozoospermia
Microdeletions of the Y chromosome (Yq) include the azoospermia factor (AZF) regions with several copies of spermatogenic genes. The high oligozoospermic rate of approximately 4% of men with sperm densities of 0.1-5 million/ml. The AZFc deletion accounts for about 60% of Y microdeletions, and of these men, one-third have severe oligozoospermia. Moreover, the high homology within palindromic sequences induces intra-chromosomal recombination and deletions of 0.8-7.7 megabase segments that arise in embryonic life [4] (McLachlan, 2013).
AZF is divided into regions a, b, and c in the Y chromosome. Complete AZF deletions rates in the general population are rare (1 in 4,000); however, they happen in 10% of patients with idiopathic nonobstructive azoospermia and 5% of men with severe oligozoospermia (mainly with a male who has <2 million spermatozoa per ml). Another factor of AZF mutation is the carriage of NAHR substrates inside the AZFc region, which can arise the development of various partial AZFc deletions. The most clinically relevant NAHR deletion is the gr/gr deletion for its frequency. NAHR deleted half of the AZFc region gene content, which resulted in a substantially increased risk of a male having oligozoospermia [5] (Krausz and Riera-Escamilla, 2018).
Moreover, constitutional chromosome irregularities are also a common cause of male infertility, detected in up to 20% of infertile men with oligozoospermia. Furthermore, another research study the correlation between prewash progressive sperm motility and pregnancy rate in severely oligoasthenozoospermic patients in (ICSI) cycle. The patients were classified into two groups depending on prewash progressive sperm motility [9] (TURHAN, PEKEL, AYRIM and BAYRAK, 2011). Group one had progressive sperm motility under 10%, and group two had progressive sperm motility equal to or greater than 10%. The result shows no significant differences among the two groups regarding the total number of oocytes retrieved, number of mature oocytes, fertilization rate, or the number of transferred embryos. In group two, the clinical pregnancy rate was significantly higher where sperm motility was higher ( (Figure 2).
Sperm parameter, such as morphology and motility impact fertilization and pregnancy rate, in addition to sperm DNA integrity, which is considered to be a major factor of the quality of the injected sperm. Male infertility with high sperm DNA fragmentation index (DFI) has result in poor ICSI outcomes with lower fertilization rate, pregnancy rate, live birth rate, and higher abortion rate (HM, 2019) [11]. HM, 2019, conducted a study to compare (ICSI) results in patients with severe oligospermia using testicular vs ejaculated spermatozoa. As testicular sperm has been shown to have considerably less DNA fragmentation than other sperm sources; as a result, the use of the testicular sperm with its higher DNA integrity can generate higher success rates in oligospermia men (HM, 2019). The clinical pregnancy percentage was significantly higher in group B, where testicular spermatozoa were injected (49.50% vs 35.57%, p=0.044*). Still, there was no significant difference in the fertilization rate between group A and B (group A 67.93%, group B 68.01%, p=0.960). Moreover, high-quality embryos were found to be higher in the testicular sperm group (HM, 2019) [11].
Testicular sperms strength relies on robust chromatin integrity since the main pathways leading to sperm DNA fragmentation are induced through sperm transport into the seminiferous tubules or epididymis transit (Esteves, Roque and Garrido, 2018) [12]. The validity of this biological aspect relies on, firstly, chromatin compaction is continuous throughout epididymal transit. Secondly, high reactive oxygen species can be produced in the epithelial cells of epididymis under physicochemical stressors like high temperature and environmental circumstances. Finally, the DNA of mature live sperm can be cleaved by endonucleases. Consequently, sperm DNA fragmentation can occur within various pathways, revealing the high rate of SDF in live ejaculated sperm ( Figure 3) (Esteves, Roque and Garrido, 2018) [12]. Therefore, testicular sperm collected through testicular sperm aspiration (TESA) or extraction (TESE) have the potential of using and selecting spermatozoa free of DNA damage for ICSI ( Figure 4). Besides, oocyte fertilization by a gnomically intact testicular spermatozoon will increase the possibilities of forming a normal embryonic genome that will eventually improve the likelihood of pregnancy and live birth (Esteves, Roque and Garrido, 2018) [14].
Furthermore, as mentioned before, differences observed in fertility outcomes in male infertility treatment are due to the sperm DNA fragmentation level between ejaculated and testicular sperm. For instance, the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) positive sperm with DNA fragmentation is lower in the testicular sample compared with ejaculated samples.  Figure 4: Overview of TESA procedure. A) Percutaneous Testicular Sperm Aspiration where a small biopsy is aspirated of the testis tissue using a needle attached to a syringe (A1, A2). In B, conventional Testicular Sperm Extraction (TESE) is performed to open scrotal layers down to the albuginea. In C1, Testicular biopsy is flushed into the sperm media tube, then the testicular biopsy is washed free from blood clots, and seminiferous tubules are mechanically dispersed (C2); finally, the testicular homogenates are examined under the microscope to prove the presence of spermatozoa (Esteves, Miyaoka and Agarwal, 2011) [13].

Conclusion :
Sever oligospermia indicate low sperm count, which can lead to male infertility; severe oligospermia be can overcome through ICSI. Genetic factors like microdeletions of the Y chromosome (Yq) can cause severe oligospermia or chemotherapy molecules, affecting the sperm count directly. The research results indicate that ICSI effectively treats severe oligospermia, mainly if testicular sperms are used through TESA. Since testicular sperms have a robust chromatin integrity, which can save sperms from DNA fragmentation in cases of sever oligospermia.