The present systemic review and meta-analysis showed that among patients with cryptozoospermia undergoing ICSI, the use of testicular sperm leads to higher good-quality embryo rate, implantation rate, and pregnancy rate in compared with ejaculated sperm.

Because the surgical extraction of sperm was adapted in ICSI, numerous studies have discussed how sperm origin affects clinical outcomes. These studies have yielded divergent results, and researchers have provided many probable theories to support their findings. Some evidence suggested that ejaculated sperm should lead to a better fertility outcome because of its passage through the epididymis. The epididymis plays a crucial role in the final steps of spermatogenesis, including epigenetic modification of genes13,14, changes in the surface proteins of spermatozoa15, and maturation of sperm cells16. Some epigenetic remodelling processes is necessary for the stability of DNA and its resistance to damage and have been reported to play a crucial role in early embryogenesis17,18,19,20,21. Alteration of these epigenomes is associated with low sperm quality, low embyro quality, and male infertility19. With regard to the role of the epididymis in sperm maturity, an immunostaining study demonstrated that the protein which facilitates the penetration of the zona pellucida by the sperm was found localised only on the acrosomal cap of epididymal sperm cells but not on testicular sperm cells22. The maturation process of sperm was also observed in IVF assays performed with spermatozoa collected at different sites along the epididymis, in which sperm retrieved from the more distal part acquired higher motility and ability to efficiently encounter the egg and its vestments16. Because testicular sperm does not undergo these modification processes in the epididymis, this may explain why some studies have shown higher normal fertilisation rates, implantation rates, and pregnancy rates23,24,25, and lower abortion rates in ICSI cycles using epididymal sperm than those using testicular sperm26,27.

However, some studies have shown no difference in outcomes of ICSI with testicular and epididymal sperm or with testicular and ejaculated sperm28,29. Some studies have even shown better fertility outcomes with testicular sperm than with ejaculated sperm4,13,30. These studies concluded that although the complex mechanisms involved in epididymal transport may be beneficial for the conventional fertilisation of oocytes, the technique of ICSI bypasses the investment and membrane of oocytes. Also, during ICSI the technician always attempts to select the sperm with highest motility and healthiest morphology through thorough examination. Therefore, the maturation of sperm is no longer a factor affecting fertilisation ability9,29. Damage to the sperm DNA along the genital tract might further explain why testicular sperm leads to better fertility outcomes than does ejaculated sperm. In the ICSI outcomes, studies have suggested that damage to sperm DNA may lead to impaired sperm decondensation, which reduces fertilisation rate31,32, produces low-quality embryos33, leads to implantation failure34, and causes recurrent pregnancy loss35,36. These findings raise the question of where the focus of sperm DNA damage lies during its passage through the genital tract. In a mouse model, sperm extracted from after the caudal epididymis exhibited higher chromatin aberrations and lower fertility compared with sperm extracted before the caput epididymis37. Furthermore, in infertile men with a high DNA fragmentation rate, testicular sperm showed less DNA damage than did ejaculated sperm38,39,40. One theory for this result is that the mature spermatozooa are tightly packed with ROS-producing immature spermatozooa while passing through the semineferous tubule and epididymis, which cause DNA damage to the mature sperm41,42. This theory is reasonable because spermatozoan DNA is more vulnerable to oxidation before it undergoes chromatin condensation in the epididymis. The result of our study suggests that the oxidative stress during passage through epididymis may pose more threat to ICSI outcome than the immaturity of testicular sperm.

Before our study, a previous systematic review with meta-analysis found 250 citation records, included four cohort studies and one case report, and gave a controversial conclusion that was based on incorrect pooling result of pregnancy rate from 272 ICSI cycles12. The previous systematic review indicated that no difference between testicular sperm group and ejaculated sperm group with forest plots for pregnancy rate (OR = 0.53, 95% CI 0.19–1.42, P = 0.21, I2 = 67%) and fertilisation rate (OR = 0.91, 95% CI 0.78–1.06, P = 0.21, I2 = 73%). These results showed moderate to high heterogeneity with less explanation and interpretation.

Our systematic review found 312 citation records, and included six cohort studies6,7,8,9,10,11. To conduct a reasonable quantitative synthesis, we excluded a case report4 that was included in the previous systematic review12. Our meta-analysis showed that male with cryptozoospermia in testicular sperm group had higher pregnancy rate than in ejaculated sperm group. There are two potential reasons for this result that is different from the previous systematic review. First, qualitative synthesis with different study design is a methodological difference between our study and the previous meta-analysis. The previous meta-analysis combined cohort studies with a case report, but our meta-analysis synthesized cohort studies only. Second, the previous meta-analysis12 reported for ejaculated sperm 9 pregnancies out of 27 attempts and for testicular sperm 5 pregnancies out of 21 attempts, while Bendickson report 9 pregnancies out 21 attempts for testicular sperm and then 5 out of 27 for testicular sperm7. We corrected this error in our meta-analysis.

Moreover, our meta-analysis supplemented important outcomes of good-quality embryo rate and implantation rate. We also conducted subgroup analyses to reduced confounding bias and heterogeneity. Therefore, a high heterogeneity was detected in subgroup that including estimated data and frozen sperm data (I2 = 81%, P = 0.006) on the result of fertilisation rate. The other subgroup that using fresh sperm data showed acceptable heterogeneity (I2 = 34%, P = 0.21). Other subgroup analyses revealed acceptable heterogeneity between I2 0% to 19% in good-quality embryo rate and pregnancy rate. There is only subgroup analysis of implantation rate that showing moderate heterogeneity. The moderate heterogeneity may be affected by some limitations of this systematic review.

Although the present systematic review has much strength that was discussed above, it has some limitations. First, we cannot separate intention-to-treat and per-protocol data for meta-analysis, because of no sufficient information in the cohort studies. This may be a critical issue for all treatment recommendations. Second, we cannot solve confounding factors from risks of testicular biopsy, because of no sufficient information. Third, it is hard to deal with frozen-thawed sperm and sperm retrieval approaches perfectly, though we have tried to do subgroup analysis. We detected high heterogeneity in subgroup that including estimated data and frozen sperm data, and reduced heterogeneity from I2 = 60% to I2 = 34% in subgroup of fresh sperm data. These results raise a question regarding whether the cryopreservation of sperm affects ICSI outcomes. In our analysis, because we could not know the exact number of patients and cycles they had undergone with fresh or frozen testicular sperm in each study, we could not accurately stratify those data to observe the relations between cryopreservation and ICSI outcomes. Meanwhile, in our review of previous studies, comparing the outcomes of fresh and frozen testicular sperm, most studies focus on patients with azoospermia rather than on patients with cryptozoospermia. Therefore, this topic necessitates further investigation. Fourth, regarding to ovarian hyperstimulation protocols, evidence had showed that different ovarian hyperstimulation protocols may have influence on the ICSI outcome. A meta-analysis has shown that the pregnancy rate was found to be higher in long protocol than short or ultrashort protocol when GnRH agonist was used43. When comparing GnRH agonist with antagonist used in ovarian hyperstimulation, there was no difference in live birth rate but a lower incidence of ovarian hypertimulation syndrome in GnRH antagonist group44. For now, the available studies are not sufficient for subgroup analysis of possible influence of different ovarian hyperstimulation protocols on the ICSI outcome. This is a topic required future investigation. Fifth, the maturity and morphology of oocyte also play a role in the ICSI outcome45. Maternal age is an important factor for oocyte quality46. In the six studies included in our study, there was a trend of higher maternal age in testicular group than ejaculated group. This could be explained by the fact that non-invasive ejaculated sperm collection was usually first used in infertile couples, and testicular sperm extraction was performed only after failure with ejaculated sperm6. Our meta-regression analysis showed that advanced maternal age was not a significant negative predictor of pregnancy outcome. However, due to the lack of information about the intracytoplasmic or extracytoplasmic features of the metaphase II oocytes in the included six studies, the relationship between oocyte quality and ICSI outcome could not be determined. Further studies are needed for subgroup analysis of effect of oocyte quality on ICSI outcome. Sixth, Surgical recovery of testicular sperm is associated with complications including vascular injury, hematoma, infection, fibrosis or even hypogonadism47. This is a reason why the included studies are all cohort studies instead of prospective randomized control trial, because it would be unethical to randomly allocate patients to TESE group without first attempting sperm retrieval with ejaculation. In these studies, the numbers of failed ejaculation attempt before conducting TESE were not reported, and whether the information about possible complications after TESE was inform to the couples were also not mentioned in these studies. Because of the lack of information about the morbidity after TESE in the six studies included in our meta-analysis, the balance between risk and benefit of TESE should be handled carefully based on future studies or experts’ experience.

In summary, the present systematic review found that the use of testicular sperm led to higher good-quality embryo rate, implantation rate, and pregnancy rate than ejaculated sperm in ICSI among patients with cryptozoospermia. Although this systematic review with meta-analysis has some limitations, it still provides more informative, reliable, and clear results than previous studies. Based on existing evidence, these results may support the recommendation for male with cryptozoospermia to use testicular sperm in preference over ejaculated sperm for ICSI.