In the present study, males with extremely severe oligospermia (MESO) were defined as having $\le$2000 motile sperm/mL of semen, while males with severe oligospermia (MSO) were defined as exhibiting 2000–10,000 motile sperm/mL. For those males, the most effective and efficient method to achieve pregnancy is in vitro fertilization (IVF) using intracytoplasmic sperm injection (ICSI) technology [1]. Extracting adequate sperm for selection and subsequent injection is crucial, particularly for MESO patients. If we fail to extract sperm from ejaculated semen samples from MESO, sperm retrieval may have to shift to the epididymis or testis [2]. The drawbacks of sperm retrieval from epididymis or testis are evident. First, these are invasive procedures that can cause discomfort and pose potential health risks to male patients [3]. Second, since surgical sperm retrieval requires a significant recovery period, it may significantly delay subsequent IVF cycles if the initial attempt is unsuccessful. Therefore, how to obtain adequate sperm from semen samples of MESO patients is particularly important.

Commonly, due to its high sperm recovery rate, density gradient centrifugation (DGC) is widely adopted for sperm preparation in reproductive centers worldwide, including ours [4, 5]. In most cases, a single DGC (SDGC) is sufficient to obtain sufficient sperm for ICSI, despite a low recovery rate. However, for MESO, the sperm recovery rate using SDGC is very low, making it challenging to find morphologically good sperm for ICSI. We previously demonstrated that double DGC (DDGC) significantly increases the sperm recovery rate from poor-quality semen samples for conventional in vitro fertilization, including those with reduced motility, poor liquefaction, or excessive clots and fibers [6]. Thus, we speculated that DDGC may be a promising strategy for improving sperm recovery rate in MESO. However, whether DDGC may enhance sperm recovery rate from MESO for use in ICSI still needs further investigation.

Another concern is that the safety of using DDGC for ICSI in MESO remains unclear. Previous studies have indicated that a second round of DGC may introduce additional mechanical stress on sperm, compromising their quality and potentially impairing both in vitro and in vivo embryo development [7, 8]. Although we previously showed the clinical safety of using sperm prepared by DDGC in combination with swim-up (SU) procedure [6], there are two main differences between that study and the present one. First, the SU procedure itself can further select sperm [9]. In the present study, however, the extremely low sperm density in MESO prevented the use of SU procedure. Second, sperm from MESO may exhibit functional defects [10]. The additional mechanical stress caused by a second DGC step may further compromise these already “fragile sperm”, potentially impairing embryo development. Therefore, the safety of using DDGC-prepared sperm from MESO without a SU procedure for ICSI remains uncertain and warrants further investigation.

In the present study, we first compared sperm recovery efficiency between the SDGC and DDGC methods using MESO samples to determine whether DDGC could effectively enrich sperm from these samples. We then retrospectively analyzed in vitro fertilization data from 39 MESO cases (sperm prepared by DDGC, DDGC group) and 78 MSO cases (sperm prepared by SDGC, SDGC group) collected between 2017 and 2023 at the Reproductive Center of Changzhou Maternal and Child Health Care Hospital to determine whether sperm processed by DDGC from MESO samples are safe for clinical use. This study may be the first to provide a routine IVF sperm preparation method for MESO.

MESO is a rare clinical condition, affecting approximately 0.6% of couples undergoing IVF at our reproductive center. Obtaining adequate motile sperm from MESO samples is crucial for the success of ICSI. In this study, we demonstrated that DDGC significantly increased the sperm recovery rate from MESO samples, meeting the requirements for ICSI. Importantly, no adverse effects on in vitro or in vivo embryo development were observed when using DDGC-prepared sperm from MESO.

In normal semen samples with adequate sperm counts, sperm cells aggregate to form a pellet during centrifugation, which accelerates sedimentation and allows enrichment using SDGC. In contrast, in semen samples from MESO, a low sperm count prevents the pellet formation, leading to a very slow sedimentation rate and ineffective enrichment by SDGC. Although SDGC is widely used for sperm enrichment, it fails to produce satisfactory results in MESO cases. Consistent with our previous study [6], the present study showed that DDGC significantly increased the sperm recovery rate from MESO samples, yielding post-purification sperm densities more than three-fold higher than pre-purification levels, providing adequate sperm for ICSI.

A potential concern is whether additional centrifugation could damage the sperm, thereby compromising the embryo developmental potential. To avoid the potential failure of sperm acquisition from MESO using SDGC, our center routinely uses DDGC for sperm enrichment in these cases. However, SDGC is still used for sperm enrichment in MSO cases at our reproductive center. Therefore, SDGC for MSO was used as the control in this study. The general condition of the couples was comparable, although the ovarian reserve was smaller in the SDGC group than in the DDGC group. Since previous studies, including ours, have reported that ovarian reserve does not affect oocyte quality [12, 13], this difference is unlikely to introduce bias. We found that the normal fertilization rate and top-quality day-3 embryo formation rate were similar between the SDGC and DDGC groups. Surprisingly, the usable blastocyst formation rate was significantly higher in the DDGC group than in the SDGC group. We observed that maternal age was lower in the DDGC group, although not statistically significant. Therefore, the higher usable blastocyst formation rate in the DDGC group may be due to younger female age. Overall, these results indicated that DDGC in MESO patients does not impair in vitro embryo developmental potential compared to SDGC in MSO patients.

It has been reported that semen quality is not associated with clinical pregnancy rates [14, 15]. However, poor semen quality has been reported to be associated with pregnancy loss [16, 17]. If the additional round of DGC truly damaged sperm in MESO samples, the DDGC group would be expected to show lower clinical pregnancy and live birth rates, along with a higher abortion rate. However, the clinical pregnancy rate, live birth rate, and abortion rate were comparable between the SDGC and DDGC groups, even after adjusting for confounding factors. Interestingly, gestational age and birth weight were higher in DDGC group for both singleton and twin births, with gestational age in twin pregnancies being significantly longer (p 0.05). One of the adverse effects of twin pregnancies is preterm delivery and low birth weight [18, 19], so increasing gestational age may benefit the health of twin births. Therefore, a longer gestational age and higher birth weight are positive outcomes. However, gestational age and live birth weight can be affected by numerous confounding factors, such as nutritional status and overall health conditions [20, 21]. In addition, the sample size is limited. Therefore, it remains uncertain whether this difference is attributed to DDGC. In conclusion, these results suggest that DDGC in MESO patients does not impair in vivo embryo developmental potential compared to SDGC in MSO patients.

Although increased mechanical stress has been reported to damage sperm organelles and membranes [7, 8, 22, 23, 24], it has also been shown that simultaneous removal of the sperm plasma membrane and acrosome before ICSI improves oocyte activation and embryonic development [25], indicating a central role of sperm DNA. We hypothesize that an additional round of centrifugation is unlikely to cause sperm DNA damage, as DNA is highly compacted within the sperm head. This hypothesis is further supported by a previous study showing that over-centrifugation of mouse sperm did not affect fertilization rates [26]. Additionally, it is reasonable to deduce that if sperm membrane integrity were compromised by the additional DGC, sperm motility would likely be affected. During ICSI, only morphologically normal sperm with good motility are selected for injection. Therefore, even if additional DGC increases the proportion of damaged sperm, the sperm selected for injection is unlikely to be damaged.

To the best of our knowledge, our study is the first to focus on sperm enrichment from MESO semen samples. Although emerging sperm preparation techniques such as microfluidic sperm sorting, electrophoretic sperm selection, and magnetic-activated cell sorting (MACS) are gaining attention [27, 28], these methods aim to isolate healthier sperm while minimizing mechanical stress, rather than improving sperm recovery rates [27]. While MACS has demonstrated recovery rates comparable to or exceeding those of conventional methods like DGC [29], its efficiency depends heavily on forward sperm progression [30], raising uncertainty about its suitability for processing MSO or MESO semen samples. Similarly, electrophoretic sperm selection has been reported to achieve only half of the recovery rate of DGC [31]. A study combining SU or DGC with MACS have shown improved sperm quality, but also a significant reduction in total sperm count and in the proportion of rapidly progressive spermatozoa [32]. Although advanced techniques may enhance sperm quality, their impact on IVF outcomes remains inconclusive [33]. Moreover, these methods are used for conventional semen samples rather than MESO, and formal studies on sperm enriching from MSO or MESO are lacking.

Another strength of the present study is that DDGC has been routinely used for sperm enrichment from MESO at our reproductive center. After implementing DDGC, concerns about failing to obtain sufficient sperm from MESO for ICSI are eliminated. In addition, DDGC does not require additional equipment and is a simple method that meets clinical requirements.

In conclusion, our study demonstrated that DDGC can effectively enrich sperm from MESO semen samples. Importantly, DDGC did not appear to compromise either in vitro or in vivo embryo development, supporting the preliminary safety and feasibility of this method. These findings hold significant clinical value as they provide a simple, efficient, and safe method for sperm enrichment for MESO. Therefore, DDGC could potentially be adopted as a routine sperm preparation method for MESO in IVF centers. Nevertheless, the long-term safety of offspring conceived via DDGC remains to be established, and the development of more effective methods for sperm enrichment from MESO is still needed.

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

XD, YW and LC designed the research study. LL, QW and XL performed the research. LL, QW, XY and YW analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All couples included in the study read and signed the informed consent forms. This study was approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital 2023[45]. All treatments were performed in strict accordance with the Declaration of Helsinki for Medical Research.

This work was supported by the Cutting-edge technology project (QY202405), and the Changzhou Municipal Health Commission Youth Science and Technology Project (QN202241).

The authors declare no conflict of interest.

During the preparation of this work, the authors used ChatGPT-3.5 to check spelling and grammar. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/CEOG43582.