Introduction

Y chromosome microdeletion is one of the major genetic factors contributing to male infertility in cases of azoospermia or severe oligospermia1. The AZF (azoospermia factor) is located on the long arm of the Y chromosome, and the absence of this region may lead to a decrease in sperm count or disordered spermatogenesis2. As recommended by the European Society of Andrology and the European Molecular Genetic Quality Control Network, the detection of Y chromosome AZF deletion is a crucial step in the diagnosis of azoospermia and severe oligospermia3. Based on different regions, AZF deficiency can be categorized into three regions—AZFa, AZFb, and AZFc4. Since the AZFc region is particularly vulnerable to non-allelic (intrachromosomal) influences, this can lead to microdeletions or duplications5,6. Clinically, we have observed that carriers of AZFc region deletion (ranging from azoospermia to normal sperm count) exhibit surprising phenotypic variations, although the reasons remain unclear.

Thanks to the development of high-throughput sequencing technology, we have been able to conduct an in-depth investigation of the AZF region and have described several different AZFc partial deletion patterns. The AZFc region can be divided into gr/gr, b2/b4, b1/b3, b1/b2, u3/g1, b1/u3, b2/b3, etc7. It is of great significance to explore whether there are significant clinical differences among different deletion patterns.

The pathogenesis of patients with idiopathic NOA remains unclear. However, deletions in the AZFc region are recognized as being significant genetic factors. Notably, the effects of various microdeletion patterns on the success rates of sperm extraction in NOA patients and the outcomes of ICSI-assisted pregnancies have not been thoroughly investigated. To address this gap, we reviewed and analyzed the clinical data of NOA patients with AZFc deletions who underwent ICSI-assisted pregnancies. According to the incidence characteristics of AZF microdeletions, there are many types of microdeletions, but the probability of their occurrence is extremely low. Therefore, based on their probability of occurrence, we specifically focused on three common deletion types, gr/gr, b2/b4, and b2/b3, to provide guidance for the development of assisted reproductive strategies for these patients.

Data and methods

Study object and group

This study employed a retrospective cohort design. It analyzed patients diagnosed with NOA who exhibited either a total or partial deletion of the AZFc region on the Y chromosome. These patients were admitted to the Reproductive Center of Henan Provincial People’s Hospital between December 2016 and September 2023 and underwent micro-TESE. Subsequently, those from whom sperm was successfully extracted received treatment through ICSI to facilitate assisted pregnancy. The study received ethical approval from the Reproductive Medicine Ethics Committee of Henan Provincial People’s Hospital (SYZZ-LL-2019110401), and the guidelines outlined in the Declaration of Helsinki were followed. All participants signed a written informed consent form.

Inclusion Criteria: (1) Patients who underwent three semen analyses, in accordance with the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition), with no sperm detected via microscopy following centrifugation; (2) patients with partial or complete deletions in the AZFc region of the Y chromosome, as identified through high-throughput sequencing, were categorized into three groups based on deletion type—gr/gr deletion, b2/b4 deletion, and b2/b3 deletion; (3) patients who underwent micro-TESE, with those successfully retrieving sperm proceeding to ICSI for assisted reproductive treatment; and (4) the patient was not exposed to an environment that significantly affected fertility, such as smoking, alcohol, drug use, etc.

Exclusion Criteria: (1) Patients exhibiting chromosomal karyotype abnormalities or single-gene disorders that necessitate preimplantation genetic diagnosis or screening; and (2) couples in which the female partner has severe ovarian dysfunction or endometrial diseases that may significantly impact the outcomes of ICSI-assisted pregnancies.

Research method

  1. 1.

    Micro-TESE: Patients who were included in the study underwent surgery according to a routine surgical plan. If sperm were found during the operation, it was considered to be positive for sperm collection, and the embryologist determined whether such a collection method was available. The evaluation of sperm during the operation was based on the description of sperm morphology in the WHO Guidelines 5/6; priority was given to sperm with normal sperm morphology. Sperm with “malformations” or “deformities” were confirmed by the embryologist as unable to undergo ICSI, i.e., sperm head vacuolation, double-headed sperm deformity, point-headed sperm deformity, big-headed sperm deformity, etc.

  2. 2.

    AZF: The sequencing library was constructed according to the instructions of the DNA extraction kit (Tiangen Biochemical Technology, Beijing, China) and the Y chromosome microdeletion detection kit (Beijing Zhongyi Health Medical Equipment Co., LTD.). High-throughput sequencing is performed using a MiSeq sequencer (Illumina, Nextseq 550 DX) according to the instructions of the general sequencing reaction kit (Beijing Zhongyi Kangwei Medical Equipment Co., LTD.). Captures were performed according to the specific probes designed, and the relative copy number of the AZF region was calculated, using the average number of autosomal housekeeper probes as a reference. Subsequently, the Z-Score method was applied to validate the preliminary findings and to determine the final variation type of the sample.

  3. 3.

    ICSI and ET (embryo transfer): The ovulation induction, ICSI, and embryo transfer protocols were developed based on the relevant routine procedures of the department. Blood samples were collected 14 days post-transfer to measure β-hCG levels, and a vaginal ultrasound was performed 28 to 35 days after the transfer. If a gestational sac is visible, clinical pregnancy is confirmed, and the number of gestational sacs is recorded. Follow-up via telephone continues until the birth of the fetus.

Statistical methods

For data analysis, measurement data consistent with a normal distribution were expressed as mean ± standard deviation (SD). Group comparisons were conducted using either the independent samples t-test or one-way analysis of variance (ANOVA), as appropriate. Qualitative data were presented as percentages (%), and group comparisons were performed using the Chi-square test or Fisher’s exact test, depending on the data characteristics. All statistical analyses were conducted using SPSS version 23.0, with a p-value of < 0.05 being considered as statistically significant.

Results

Overview of the basic situation and comparison of SRR

According to the established inclusion and exclusion criteria, a total of 90 patients with deletions in the AZFc region associated with NOA were included in this study. In total, 50 micro-TESEs resulted in sperm, yielding an SRR of 55.6% (50/90). The number of micro-TESE procedures with sperm that had severe morphological abnormalities, which is deemed unsuitable for ICSI in assisted reproduction, was 13. Consequently, the number of micro-TESEs that resulted in available sperm was 37, translating to an available sperm retrieval rate of 41.1%.

As illustrated in Table 1, the SRR in the gr/gr deletion group was significantly lower than that in the b2/b4 deletion group, at a statistically significant level (P < 0.05). The unusable malformed sperm were derived from the b2/b4 deletion group. Additionally, the volume of the left testicle in the b2/b3 deletion group was smaller than that in the b2/b4 deletion group, and the follicle stimulating hormone (FSH) levels were significantly higher in the b2/b3 group compared to the other two groups (P < 0.05). the luteinizing hormone (LH) levels were significantly higher in the b2/b3 group compared to the other two groups (P < 0.05). No statistically significant differences were observed in age, volume of the right testicle, prolactin (PRL), testosterone (T), or estradiol (E2) levels among all groups (P > 0.05).

Table 1 Comparison of the general situation among patients with deletions in different parts of the AZFc region.
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Table 2 Comparison of the general outcomes of couples undergoing ICSI treatment in patients with various extents of AZFc deletion.
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Comparison of embryo development and embryo transfer outcomes assisted by ICSI

A total of 37 couples with available sperm for ICSI-assisted pregnancy were included in the study. As presented in Table 2, the overall characteristics of both partners in each group exhibited no statistically significant differences in any indicators (P > 0.05).

The development of embryos in ICSI cycles across various AZFc deletion groups was compared. The number of micro-TESEs that resulted in available sperm was 37, and 52 oocyte retrieval cycles were performed. As illustrated in Table 3, there were no significant differences among the groups in terms of the total number of oocytes collected, the number of MII oocytes, the fertilized oocyte (2PN) rate, the 2PN cleavage rate, the D3 available embryo rate, the high-quality embryo rate, and the blastocyst formation rate (P > 0.05).

In our comparative analysis of embryo transfer outcomes within ICSI cycles across distinct AZFc deletion subgroups, we examined a total of 57 transfer cycles distributed among the three groups. Our findings, as detailed in Table 3, indicate that there were no significant variations in the number of transferred embryos, live birth delivery rate, clinical pregnancy rate, or miscarriage rate (P > 0.05). However, the implantation rate for the gr/gr deletion group was notably higher than that observed in the other two groups; this is a difference that was statistically significant (P < 0.05).

Table 3 Comparison of ICSI embryo development and embryo transfer outcomes among various AZFc deletion categories.
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Discussion

The AZFc region of the Y chromosome spans approximately 4.5 megabases (Mb) and harbors critical genes, including DAZ and CDY1, which are closely associated with male spermatogenesis and sperm morphology. Notably, the presence of multiple gene copies within this region means that partial deletions of AZFc may not necessarily result in the complete loss of these genes8,9,10. Advances in high-throughput sequencing technology have enabled the classification of AZFc into distinct subtypes, such as gr/gr, b2/b4, b1/b3, b1/b2, u3/g1, b1/u3, and b2/b3. Among these, the b2/b4 deletion is considered equivalent to the complete deletion of the AZFc region. Research in murine models has demonstrated that deletions in the long arm of the Y chromosome (MSYq) lead to varying degrees of spermatogenic impairment, ranging from abnormal sperm morphology to complete infertility11. Furthermore, evidence suggests that the DAZ gene family within the AZFc region plays a pivotal role in the regulation of sperm morphology, with its dysfunction being linked to morphological abnormalities12.

The gr/gr deletion subtype is characterized by a partial deletion that primarily reduces the copy number of the DAZ, CDY1, and BPY2 genes. Patients with this type of deletion exhibit significant phenotypic variation, ranging from azoospermia to normal sperm parameters. Currently, the clinical significance of the gr/gr deletion remains a topic of debate. Some studies suggest that the functional differences in AZFc gene copies among various populations may contribute to the inconsistent relationship between gr/gr deletion and spermatogenic dysfunction13. Conversely, other studies have supported the notion that gr/gr deletion is a risk factor for spermatogenic dysfunction3.

The b2/b3 deletion, which also represents a partial deletion, primarily results in the deletion of DAZ1 and DAZ2, distinguishing it from the gr/gr deletion14. The differentiation between these deletions may influence the regulatory roles of other non-coding regions, leading to varying clinical implications. However, the effects of different AZFc region subtypes on SRR and outcomes of ICSI have yet to be reported.

This study is the first to compare the SRR of patients with NOA who have different deletions in the AZFc region and who underwent micro-TESE. The overall SRR for patients with AZFc region deletions in this study was 55.6% (50/90), which aligns closely with findings reported by Mao Jiaming et al.15. According to our classification of deletion types, we found that patients with a complete deletion of the b2/b4 region exhibited the highest SRR at 64.5% (40/62). However, patients with partial deletions demonstrated severe sperm malformation [n = 13, accounting for 32.5% (13/40)] and the inability to provide sperm suitable for ICSI. The SRR for the gr/gr deletion group was 23.5% (4/17). This rate is significantly lower than that of the b2/b4 deletion group. The SRR for the b2/b4 deletion group was similar to that of the b2/b3 deletion group, which had an SRR of 54.5% (6/11). There was no statistically significant difference in the right testicle volume of patients in each group, but the left testicle volume of the b2/b3 deletion group was smaller than that of the other groups, that is, the total testicle volume of the b2/b3 deletion group showed a downward trend compared with other groups. In our study, the FSH levels and the LH levels in the b2/b3 deletion group were significantly higher than those in the other groups, which may provide clues to the mechanism of impaired spermatogenesis function caused by this type of deletion, and we can further explore the causes of this correlation in subsequent studies.

Based on the above data, we conclude that microdeletions in different regions of the AZFc region have varying impacts on spermatogenesis in patients with NOA. The deletion of the gr/gr gene has a significant impact on the SRR of micro-TESE, which is comparable to that observed in cases of idiopathic NOA16,17,18. Therefore, we boldly hypothesize that gr/gr deletion may not be the cause of NOA in such patients. Some researchers have suggested that microdeletions in the AZFc region may not occur in isolation; rather, they may coincide with the activation of other genes and influence spermatogenesis through mechanisms such as gene duplication or dosage compensation. This interaction could mitigate the effects of microdeletion on spermatogenesis, leading to variations in clinical phenotype11. Therefore, while the precise mechanisms underlying these differences remain unclear, the findings of this study carry significant implications for developing clinical strategies for affected patients.

The impact of the AZFc microdeletion on assisted reproductive outcomes, particularly in the context of ICSI, has garnered significant attention in recent studies. Research has primarily focused on the sources of sperm, specifically comparing ejaculated sperm and sperm obtained through testicular biopsy (testicular sperm extraction—TESE). Previous investigations have indicated that AZFc microdeletion negatively affects ICSI outcomes. Specifically, when compared to a control group without Y chromosome deletions, patients with AZFc microdeletions exhibit reduced clinical cumulative pregnancy rates, cumulative live birth delivery rates, embryo transfer suitability rates, fertilization rates, and implantation rates19. A recent meta-analysis encompassing 12 studies further substantiated these findings, revealing a significant decrease in fertilization rates among patients with AZFc microdeletions. However, other parameters, including embryo quality, clinical pregnancy rates, miscarriage rates, and live birth delivery rates, were found to be comparable between these patients and those without genetic abnormalities, regardless of whether sperm was obtained via ejaculation or TESE20. For patients with NOA due to AZFc deletions, micro-TESE extraction combined with ICSI presents a viable option for achieving pregnancy and producing biological offspring, particularly when spermatogenic function is preserved. This study is the first to report on the differences in ICSI outcomes among NOA patients with distinct AZFc deletion subtypes. Our findings reveal that sperm successfully retrieved from NOA patients with AZFc deletions exhibited comparable embryo development and transfer outcomes across all groups, with no significant differences observed. However, the embryo implantation rate was notably higher in the gr/gr deletion group compared to the other two groups. Despite this, AZFc deletions—whether in patients with ejaculated sperm, testicular sperm, or NOA testicular sperm—did not significantly influence the final pregnancy outcomes. Furthermore, the specific region of AZFc deletion had no substantial impact on embryo development or pregnancy outcomes during ICSI-assisted reproduction. These preliminary results suggest that AZFc deletions, irrespective of their subtype, do not adversely affect the overall success of ICSI in achieving pregnancy.

In summary, this study represents the first comparative analysis of the SRR in NOA patients with deletions in various segments of the AZFc region, as well as an examination of the ICSI outcomes for sperm obtained from these patients. Our findings indicate that the gr/gr deletion has the most significant impact on micro-TESE among patients with AZFc deletions. In patients with b2/b4 deletions, although the high incidence of sperm malformation is found, SRR is high.Although there is no statistical difference in SRR between the b2/b3 deletion group and the gr/gr deletion group, it can also be seen that the SRR is increased.Embryo development or pregnancy outcomes following ICSI treatment are not significantly affected by all types of deletions. Therefore, patients with b2/b4 deletion and b2/b3 deletion type should actively undergo surgical treatment to obtain biological descendants.It is important to note that the prevalence of these small deletions is quite low, particularly within the NOA patient population. Only a limited number of cases were included in this study; therefore, future research should consider multi-center collaborations to enhance the sample sizes in order for more comprehensive comparative analyses to be conductedocc.