Detection of partial and/or complete Y chromosome microdeletions of azoospermia factor a (AZFa) sub‐region in infertile Iraqi patients with azoospermia and severe oligozoospermia

Abstract Background This study aimed to analyze the incidence of azoospermia factor a (AZFa) microdeletions in the Y chromosome and their association with male infertility in a population with azoospermia and severe oligozoospermia from Iraq. Methods A total of 75 infertile Iraqi males and 25 healthy controls were included in this study. The semen analysis was performed to determine the azoospermia, severe oligozoospermia, or normal cases. The AZFa microdeletions were investigated using the real‐time polymerase chain reaction (real‐time PCR). Then, AZFa sub‐region deletions were investigated by a conventional PCR. Results In total, 40 men with azoospermia and 35 men with severe oligozoospermia were selected. Out of 75 infertile males, 46 (61.3%) individuals had AZFa microdeletions, of whom 32 (69.6%) had partial deletion, while 14 (30.4%) males had complete deletion using real‐time PCR. The frequency of microdeletions was significantly different between the infertile and control group (p‐value < 0.00001). The proportion of AZFa microdeletions appeared higher in azoospermia men (72.5%, n = 29/40) than severe oligozoospermia men (48.6%, n = 17/35), but based on the conventional PCR results, only one azoospermia patient (2.2%) was shown to have complete AZFa deletion, while the other 45 patients (97.8%) had partial AZFa deletions. Conclusion In this study, the partial AZFa microdeletions were more numerous than complete AZFa deletion. According to our results, the AZFa microdeletions might be associated with male infertility and spermatogenic failure. It is recommended to investigate the AZFa sub‐region microdeletions in patients that shown AZFa microdeletions in primary screening.


| INTRODUC TI ON
Tiepolo and Zuffardi 1 were the first to identify correlations between Y chromosome deletions and male infertility. The Y chromosome is a sex chromosome and is one of the shortest human genome chromosomes (~50 million bp), representing about 2%-3% of the haploid genome. The human Y chromosome is recognized as a pauper gene, but it plays an important rᴏle.in human reproduction as its presence or absence determines gonadal sex. The Y chromosome often plays a major role in the control of sperm formation and male infertility.
It occupies a prime place in the human genome because of its organization, and job. The cytogenetic design of the Y chromosome is acrocentric, and it has a small arm (known as Yp), and a long arm (known as Yq), clearly differentiated by the centromere-region, that is necessary for chromosomal separation in male meiosis.
The human Y chromosome is essential for human sex determination and male germ cell development and maintenance. 2 The azoospermia factor (AZF) region is located ᴏn the long arm ᴏf the Y chromosome Yq. It plays a vital role in the genetics ᴏf male infertility and is divided into three sub-regions: AZFa, AZFb, and AZFc.
These regions contain genes that are involved in spermatogenesis and the development ᴏf testes. Microdeletions in this region lead to spermatogenetic defects and male infertility. 3 The AZFa sub-region is located in the proximal region in the long arm ᴏf the Y chromosome Yq. Four various genes have been recognized in this region, including ubiquitin-specific peptidase 9 Y (USP9Y) chromosome gene, previously known as DFFRY or Drosophila fat facets related Y gene; DEAD box RNA helicases, Box 3, Y-linked (DDX3Y or DBY); ubiquitously transcribed tetratricopeptide repeat containing, Ylinked (UTY); and thymosin beta 4 Y-linked (TB4Y) gene. The main gene of the AZFa region is DDX3Y, which is expressed in the testis and is involved in the progression of pre-meiotic germ cells, indicating that inhibiting DDX3Y expression plays a role in prenatal germ cell depletion and hence infertility. The USP9Y gene also assists in spermatogenesis. 4,5 Azoospermia, oligozoospermia, and oligoasthenozoospermia are caused by shortening or loss of the USP9Y gene. 6 Generally, removals of the AZFa region that delete both of these genes result in sertoli cell-only syndrome (SCOS), a state characterized not only by the presence of full sertoli cells in the testes but also by the absence of spermatozoa and azoospermia. 7 However, limited AZFa removals are correlated with phenotypes ranging from azoospermia to normozoospermia. 8 Thus, a diagnosis of total deletion of the AZFa area means that it is virtually impossible for testicular sperm through intra-cytoplasmic sperm injections (ICSI).
In a recent study by Al-Janabi et al. 9 from Iraq, the most predominant deleted region was AZFb (33.3%), followed by AZFc region (23.0%), while no microdeletion was noticed in AZFa. In severe oligozoospermia and azoospermia men, the incidence of the microdeletions in AZFa, AZFb, and AZFc regions ranged from 1% to 50.0%. In around 15.0% of couples who want to have a child, infertility is a public health concern; the male gender is present in approximately 50.0% of instances. 9 So far, few studies have been conducted on the prevalence of AZFa microdeletions in azoospermia and severe oligozoospermia infertile Iraqi patients.
Hence, this study aimed to analyze the incidence of AZFa microdeletions in the Y chromosome in patients with azoospermia and severe oligozoospermia from Iraq.

| Study patients and controls
We looked at Iraqi infertile males with spermatogenesis deficiency who had requested sterility consultations in private infertility clinics. The participants with non-obstructive azoospermia or severe oligozoospermia were selected. The participants with primary or secondary infertility were enrolled in this study. The term primary infertility used to describe infertility after one year of unprotected sexual contact in couples trying to conceive who have never before been pregnant, while secondary infertility refers to an inability to conceive after a previous pregnancy. A control group of the normal fertile males with a normal spermatogenesis was included in the study. Healthy donors and randomly selected persons were collected from the community. All exclusion criteria are applied to confirm their suitability for this group. There were no signs of gynecomastia. They were married and have offspring and normal seminal parameters. They were healthy fertile men. The selection of azoospermia, severe oligozoospermia patients, and control participants was performed according to the sperm count values stated by the World Health Organization's usual normative standards. 10 Based on these standards, absence of sperms, sperm count less than 5 million sperms/ml, and sperm count more than 15 million sperms/ml were defined as azoospermia, severe oligozoospermia, and normal state, respectively. Also, the history of continuous psychological stress and medicinal drug consumption was investigated among the studied groups.

| Semen analysis
In brief, each research participant's sperm ejaculate was acquired via masturbation at least three days of abstinence. The semen samples were centrifuged for 10 min at 1000 g. Finally, a phase-contrast microscope was used to analyze 20 µl of each sperm sample participate by a special counting chamber.

| DNA extraction
The SaMag-12 Automated Nucleic Acids Extraction System (Sacace Biotechnologies, Italy) was used to obtain genomic DNA from 1-ml whole blood samples in accordance with the manufacturer's instructions. 11,12 2.4.2 | Screening of AZFa microdeletions by multiplex real-time polymerase chain reaction (realtime PCR) assay In multiplex real-time PCR, the assay was done in two multiplex reactions (Sacace Biotechnologies, Italy) according to the European Academy of Andrology/European Molecular Genetics Quality Network (EAA/EMQN). 13 All samples were investigated for AZFa gene cluster deletion using sequence-tagged sites (STSs) markers sY84 and sY86 ( Table 1). The SRY (sex determining region on Y) and ZFY (Zinc-Finger Y) gene-specific primers were used as the internal controls, and there are two reaction: reaction A consisted of (sY86 STSs, and ZFY/X as an internal control) and reaction B consisted of (sY84 STS, and SRY as an internal control) Based on the recommendations of the EAA/ EMQN, the criteria of complete AZFa deletion in real-time PCR method are depend on the deletion of both sequence tag sites STS sY86 and sY84, while partial deletion is the lack of one and presence of another. 13 The whole reaction volume of each mul-

| Screening of AZFa sub-region microdeletions by conventional PCR
All the patients who were diagnosed with AZFa microdeletion by classical real-time PCR technique were tested with additional primers (sY82, sY88, sY1064, and sY1182) by conventional PCR to decide whether the microdeletion was partial or complete. Based on the EAA/EMQN recommendations, the criteria of complete AZFa deletion in PCR method were depend on the presence of sY82 for the start sub-region of AZFa and sY88 for the end AZFa sub-region, while deletion of the both

TA B L E 1 STS sequence of the multiplex real-time PCR and PCR primers for AZFa gene microdeletion
sY1065 for the part near to the start sub-region (proximal part) and sY1182 for the part near to the end region (distal part). The partial deletion was the presence of both the sY82 and sY88 and deletion of one of sY1065 or sY1182. 13 The primers were requested and supplied by Alpha DNA Company, Canada, in a lyophilized form, which was dissolved with sterile distilled water to give the final concentration of each primer in 100 pmᴏl/μl (

| Preparation of agarose gel with ethidium bromide
The PCR amplicons were identified using agarose gel electrophoresis and then viewed with an ultraviolet transilluminator to reveal the spe- The electrophoresis was then carried out with the following conditions: 5 volt/cm, 100 watts, for 75 min. After the electrophoresis was completed, the gel was put on a UV transilluminator. Finally, a digital picture was made for the evaluation and documentation of the results.

| Statistical analysis
The SPSS statistical software (statistical package for the social sci-  Table 2). The severe oligozoospermia cases were significantly higher than azoospermia cases in secondary infertile group, while in the primary infertile group, the opposite was observed. There was a significant correlation between the infertility types and the study groups, based on the Crosstab chi-Squared tests (χ 2 = 111.729), pvalue < 0.001. Also, there was a significant difference between the infertile and control groups in term of the presence of continuously psychological stress and history of medicinal drug consumption (Table 2). These two items were existed in more than 50% of infertile males, while no one of participants in the control group had them.

| AZFa microdeletions using real-time PCR
The results of AZFa microdeletions using real-time PCR are summarized in Table 3

| AZFa sub-region microdeletions using conventional PCR
This part of the study involved 46 patients including both azoospermic and severely oligospermic men who had previously been  (Table 4).

| DISCUSS ION
Y chromosome microdeletion in the AZF region is a common genetic source of infertility in males that may take place in three different sub-regions AZFa, AZFb, and AZFc. 3   The present study has been able to include more information about the occurrence of AZFa in Iraq, which was rarely documented previously. In our region, we found a much higher frequency of AZFa and Hanoon et al. 24 from Iraq, the AZFa microdeletions were not detected in any patients that was in contrast with the current research. These studies reported the AZFc as the most prevalent microdeletion in Iraq followed by AZFb. 9,24 Also, in a previous study from Iran, no AZFa microdeletion was identified. 25 However, in a study from Sudan, the most frequent microdeletion was found in the AZFa region (11 out of 30 patients, 71.4%), followed by the AZFc (n = 4) and the AZFb (n = 3). 26  In this study, the prevalence of AZFa microdeletions was higher in azoospermia patients than those with severe oligozoospermia.

TA B L E 3 Proportion of AZFa microdeletions in the studied groups using real-time PCR
These findings were in agreement with the findings of Dutta et al. 27 and in contrast with the results of Elsaid et al. 26 Also, in line with our findings, Sha et al. 28  Iran, 30 and Korea, 31 it was identified only in the azoospermia patients, which was contrary to this research.
This study found that infertile individuals had significantly more microdeletions than control individuals (p-value = 0.00001), suggesting that these microdeletions are associated with infertility.
These findings were in line with previous studies from Sudan 26  This study had several limitations as follows: due to limited bud- were not assayed. Moreover, the karyotyping and evaluation of testicular biopsies were not performed.

| CON CLUS ION
This study showed a high frequency of AZFa microdeletions in Iraq compared with several countries. In our study population, partial AZFa microdeletions were higher than complete AZFa deletion. We needed to study each sub-region in detail by using many primers, not only the STS primers recommended by EAA/EMQN, to judge whether the type of deletion was complete or partial. The results of this study indicated the necessity of Y chromosome microdeletion screenings for male infertility diagnosis in Iraq. For assisted reproductive techniques, it is crucial to obtain accurate genetic information to minimize the unnecessary treatments and the vertical inheritance of genetic defects to the next generation.

ACK N OWLED G M ENTS
We are grateful to the Andrology doctors for sample collection and to the staff of the Dar Al-Shefaa Medical Laboratory for their support with some experimental protocols. There is no financial support for this study.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.