Title: Optimum identification of round spermatid in men with non‐obstructive azoospermia: A commentary
Abstract: Barda et al. recently published a timely analysis regarding the utility of round spermatid injections (ROSI) in men with non-obstructive azoospermia (NOA).1 This comes in the wake of the first large-scale study documenting the extended follow-up of a Japanese ROSI clinical trial.2, 3 ROSI has now garnered significant worldwide interest, and the first ROSI clinical trial in the United States has been started.4 ROSI is an appealing option to those men who are not candidates for intracytoplasmic sperm injection due to the absence of mature spermatozoa in either ejaculate or the testes. The findings of Barda et al. directly challenge the applicability of ROSI for these patients by presenting the rate of round spermatid identification among 194 consecutive NOA patients with negative testicular sperm extraction (TESE) to be 2%. In the previous study, Tanaka et al.2 has cited round spermatid retrieval rates of roughly 30% in men with non-obstructive azoospermia. However, in the same manuscript, Tanaka et al. reported round spermatid retrieval in 76/730 men (10.4%). This study (2%) still serves as a drastic contrast to either of these values (10% or 30%), and future studies would help clarify the difference in rates. Barda et al. identified the target subpopulation of men with no spermatozoa found on TESE from a larger group of 457 consecutive azoospermic patients undergoing TESE. The histologic exam was performed on Bouin's fixed tissue following hematoxylin and eosin staining. Five different levels of spermatogenesis were described: normal spermatogenesis, mixed atrophy, spermatocyte maturation arrest at primary spermatocyte level, round spermatid maturation arrest, and Sertoli cells only. A minimum of 20 seminiferous tubules were assessed for each of the men. The histological classification was performed twice by their andrology laboratory and once by an outside pathology laboratory. Of the entire cohort, 342 were determined to have NOA, 194 of 342 (57%) had no spermatozoa, and four of 194 (2%) had round spermatids maturation arrest. Notably, as corroborated by Tanka et al., there is a level of difficulty associated with visually identifying round spermatids based solely on morphology from other round cells in the seminiferous tubules.2, 3 The authors note as much, with the other similar appearing round cells including diploid spermatocytes, spermatogonia, or potentially somatic cells potentially confused. An important issue that arises from the potential confusion of other round cells for round spermatids in the process of ROSI is a recently documented case of mistaken diploid spermatogonial fertilization that is thought to have resulted in a complete molar pregnancy requiring chemotherapy for resolution.5 An ideal solution to the selectivity of round spermatid identification in this paper would include immunohistochemistry for round spermatid-specific markers. This could be performed with specific markers such as cAMP responsive element modulator (CREM) and protamine-1 (PRM1) marker. The authors did not specify beyond the minimum number of tubules (20) how many tubules were counted on average for the NOA patients in this study. Evaluation of as low as 20 tubules may fail to assess for the presence of round spermatids accurately. To improve the sensitivity of round spermatid detection for further study, we recommend Immunofluorescence staining for specific markers of round spermatids and increasing the minimum number of examined tubules. In addition, for ultimate clinical applicability, the biopsied tissue during TESE should be frozen and then digested, similar to the process used for ROSI. Depending on the laboratory setting, a quantitative or digital reverse transcriptase-polymerase chain reaction of mixed cellular suspension, would, in theory, perform a more sensitive search of the obtained testicular tissue to determine the presence of any round spermatid markers. Supposing the techniques mentioned above succeed in demonstrating the presence of round spermatids in a larger percentage of patients than the 2% found by Barda et al., then, a process for optimizing round spermatid selection is still needed. Further study of methods such as gradient separation by cellular size and weight commonly used for sperm isolation in the in vitro fertilization clinics may offer assistance in this regard. It is worth noting that while Barda et al. focused on adult males with NOA and the retrieval rates in this population, there is an additional application for peri-pubertal or pre-pubertal males who are at risk of infertility. A recent paper published by Abdelaal et al.6 outlined the potential for fertility in pediatric male cancer patients to cryopreserve spermatogonial stem cells before gonadotoxic chemotherapy. If the spermatogonial stem cells can be differentiated in vitro into the round spermatids or if round spermatids are retrieved in peri-pubertal males at the time of cryopreservation, ROSI may theoretically be performed later in life when fatherhood is desired. The clinical application is thus broader than that described in this paper alone. Although ROSI is still experimental, it represents a potential assisted reproduction option for men with NOA and sperm-negative TESE. For some couples, the chance to conceive the biological offspring of both parents is worth the significant value despite its low success rate. This article was supported by internal findings from the Wake Forest Institute for Regenerative Medicine (WFIRM) and Carolina Fertility Institute (CFI). The authors declare that they have no conflict of interest. All authors contributed equally on writing and finalizing this manuscript.