This study, based on a large cohort of men with infertility in China, investigated the incidence of Y chromosome microdeletions and ICSI outcomes in patients with AZFcd deletions. Our results underline the necessity for detecting Y chromosome microdeletions in men with infertility and severe sperm count abnormalities, especially those with cryptozoospermia. The co-deletion of sY254 and sY255 in AZFc and sY152 in AZFd was the most prevalent pattern of Y chromosome microdeletion observed. Patients with AZFcd deletions (combination of sY254, sY255, and sY152 deletions) using ejaculated sperm and without Y chromosome microdeletions achieved similar ICSI outcomes.

Several studies have reported the incidence of Y chromosome microdeletions in Chinese populations; however, they only enrolled men with oligozoospermia and azoospermia [8, 10, 21, 22]. To our knowledge, this study is the largest epidemiological investigation of Y chromosome microdeletions and the first to report their incidence (3.13%) in Chinese men with infertility. One novel finding is that the Y chromosome microdeletions incidence among Chinese men with infertility with normal sperm counts is 0.17%, lower than the 2% incidence reported in normal men by the American Society of Reproductive Medicine Practice [23].

We detected AZF deletion rates in patients with mild oligozoospermia, severe oligozoospermia, and azoospermia of 1.13% (8/703), 5.53% (34/615), and 7.54% (70/928), respectively (slightly lower than the reported incidence of Y chromosome microdeletion in oligozoospermia [3.87–13.2%] and azoospermia [11.49–17.8%] in Chinese men with infertility, where oligozoospermia was defined based on sperm concentration) [8, 10, 21, 22]. However, the fifth edition of the World Health Organization laboratory manual for the examination and processing of human semen emphasizes that the total sperm number per ejaculate has a higher diagnostic value than sperm concentration [24]. Therefore, we utilized total sperm number to define oligozoospermia (total sperm number < 39 × 106 per ejaculate).

This study identified variations in the incidence of Y chromosome microdeletions in patients with different phenotypes of total sperm number. The incidence of Y chromosome microdeletions in patients with cryptozoospermia was 71.43%, higher than that reported in previous studies (5–11.5%) [25, 26]. This study has two implications. First, screening for Y chromosome microdeletions among patients with cryptozoospermia may be cost-effective. Second, the sperm-discovering method through semen centrifugation should be regarded, as it can help patients with suspected azoospermia determine whether they have cryptozoospermia, which has a high probability of detecting a Y chromosome microdeletion. We observed a high detection rate of Y chromosome microdeletions in Chinese men with infertility having severe oligozoospermia (total sperm number < 10 × 106 per ejaculate), cryptozoospermia, and azoospermia, suggesting that Y chromosome microdeletion testing should be considered when the total sperm number is below 10 × 106 per ejaculate.

Kent-First et al. first proposed the AZFd region in 1999, situating it between the AZFb and AZFc regions (Fig. 1) [4]. However, the AZFd region’s clinical implications remain unclear. The EAA/EMQN best practice guidelines for the molecular diagnosis of Y chromosomal microdeletions do not recommend testing AZFd regions, as the sequence of the male-specific Y chromosome region and the mechanism underlying these microdeletions indicate its nonexistence [3]. However, the AZFd region could be related in spermatogenesis and recurrent pregnancy loss [27, 28]. The guidelines and expert consensus for diagnosing and treating andrological diseases in China recommend testing AZFd regions (sY145 and sY152) [29]. Therefore, we explored the AZFd region deletion incidence and its relationship with the total sperm count phenotype. We observed that all AZFc deletions were accompanied by SY152 deletion, the predominant deletion pattern in Chinese individuals with infertility, and that SY145 in AZFd may be associated with azoospermia. When sY145 was present, the semen parameters of the AZFcd deletion group were polymorphic, ranging from normal to oligozoospermia, cryptozoospermia, and azoospermia. However, when sY145 was deleted, the patients had only azoospermia, hence the importance of detecting SY145, a gene locus, as it may contain important genes associated with spermatogenesis. More case studies are needed to confirm our AZFd regions’ observations.

Compared with patients without Y chromosome microdeletion, those with AZFc deletions can achieve similar ICSI outcomes using ejaculated sperm, but worse ICSI outcomes using testicular sperm [12, 17, 30, 31]. Studies exploring ICSI outcomes in patients with combined deletions of STS sites in the AZFc and AZFd regions are lacking. Our study suggests that patients with AZFcd deletions (combination of sY254, sY255, and sY152 deletions) using ejaculated sperm could achieve ICSI outcomes similar to those in patients without Y chromosome microdeletions, consistent with most previous studies involving patients with AZFc deletions [12, 31]. Basic data such as female BMI, female hormones, and polycystic ovary syndrome in the ovulation cycle couples to match the control group was used, reducing potential bias. Unfortunately, we did not investigate the ICSI outcome using testicular sperm in patients with AZFcd deletions owing to the small number of patients who underwent testicular sperm extraction surgery.

Usually, the relationship between age and male semen parameters change (total sperm number and concentration) after 34 years of age [32, 33]. Stone et al. analyzed semen data from 5,081 men aged 16.5–72.3 years and observed a decrease in the total sperm number after 34 years of age [32]. In a study involving 71,623 Chinese men with infertility, the critical age for the decrease in total sperm count was 42 years [33]. However, the variation pattern of total sperm count with age in patients with AZFcd deletions remains unclear. Among patients with azoospermia, oligozoospermia, cryptozoospermia and AZFcd deletion, those over 29 years had the highest proportion. Our study could not conclusively determine whether patients with AZFcd region deletion experience an earlier decline in total sperm number than those without Y chromosome microdeletion owing to the limited number of patients with AZFcd deletion (107 patients). Thus, future studies with larger sample sizes are required.

This study had several advantages. First, this is the largest epidemiological study on Y chromosome microdeletions in Chinese men with infertility, presenting the incidence of Y chromosome microdeletion in Chinese population with normal sperm count (0.17%) for the first time. Second, we discovered that the most prevalent pattern of Y chromosome microdeletion is the co-deletion of sY254 and sY255 in AZFc and sY152 in AZFd in Chinese men with infertility (68.59%[107/156] of all Y chromosome microdeletions). Third, we reported for the first time the high incidence of Y chromosome microdeletions in the Chinese population with cryptozoospermia, comprising combined deletions of sY254, sY255, and sY152; thus emphasizing the recommendation for Y chromosome microdeletion screening for patients with cryptozoospermia. If the combined deletion of sY254, sY255, and sY152 is detected in patients suspected of azoospermia, sperm should be detected using repeated semen centrifugation to avoid misdiagnosis. Fourth, we provided a reference for comprehending the clinical significance and detection relevance of AZFd deletions. The absence of SY145 in AZFd was closely associated with azoospermia, suggesting this region’s involvement in spermatogenic functions. Fifth, we are the first to explore ICSI outcomes in patients with combined deletion of STS sites in the AZFc and AZFd regions.

This study had some limitations. First, we did not distinguish between non-obstructive and obstructive azoospermia because some of these patients did not undergo ultrasound examination of the genital system. Second, we performed propensity score matching to match the basic characteristics of the AZFcd deletion and control groups. However, this method cannot eliminate potential biases. Third, this study used a small sample size to explore the effect of AZFcd deletions on ICSI outcomes. A larger sample size may be required to further substantiate our findings. Fourth, this research was conducted exclusively within the Chinese population; therefore, its generalizability to other populations may be limited. Furthermore, this study should be replicated in larger cohorts and different populations. Fifth, this study only grouped the enrolled patients based on sperm count, and did not discuss the influence of Y chromosome microdeletions on sperm motility and morphology. Sixth, the EAA/EMQN best practice Guidelines provide a clear flowchart for handling inconsistencies in genetic markers such as AZFa (sY84, sY86), AZFb (sY127, sY134), and AZFc (sY254, sY255) [3]. These guidelines recommend repeating the test in single-plex or adjusting PCR conditions to rule out potential artificial PCR results in cases of marker inconsistency. When both markers are reported as deleted, testing additional markers to confirm the complete deletion in that region is advised. Regrettably, we did not follow these recommendations for cases where only one marker was deleted in the AZFa region, nor did we conduct extension analysis when both markers were missing in an AZF region. These deviations from the EAA/EMQN guidelines may have impacted the accuracy of our study. Going forward, similar studies must adhere to the EAA/EMQN guidelines for reliable and consistent results.