LETTER TO THE EDITOR REPLY OF THE AUTHORS: We welcome the comments of Dr. Baka on our findings that were reported in ‘‘Microbiota of the seminal fluid from healthy and infertile men’’ (1) and agree that further research is warranted to understand whether any of the bacterial populations found in semen from healthy men can adversely affect male fertility potential. In our study we found there were high numbers of diverse kinds of bacteria present in most samples of both sperm donors and infertility patients. The species composition of bacterial communities varied widely among subjects, but they could be clustered into six groups on the basis of similarities in composition and the rank abundances of taxa. Dr. Baka remarked that in her opinion it was quite unusual for most of the donor sperm samples (28 of 30) to have high numbers of bacteria that outnumber sperm. In previous studies methods that require culturing of bacteria have been used to characterize bacteria of seminal fluid, and these are limited because many species of bacteria are recalcitrant to cultivation. However, in our study we used molecular methods based on the phylogenetic classification of 16S ribosomal RNA gene sequences recovered from semen samples, and this obviates the need to cultivate the bacteria present in samples. This methodology is increasingly used in studies of the human microbiome because it overcomes the inherent limitations of cultivation-dependent methods and provides a more complete and comprehensive understanding of microbial diversity. Many of the bacterial populations we observed in semen samples are strict anaerobes that are likely difficult to culture without special efforts, and we suspect this accounts for why they have been overlooked in the past. We have no reason to suspect that donors were infected or that semen samples were contaminated during the process of collection and storage. Semen donors satisfied a series of enrollment criteria that included a physical examination and sperm function tests that were done to exclude individ-

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uals with disease or subfertile subjects. Moreover, semen samples were collected according to established protocols that reduce the possibility of contamination. Dr. Baka suggested that perhaps in the future studies might be done to determine whether treating these patients with antimicrobials might improve sperm parameters and improve the outcomes of fertility treatments. Perhaps this is so, but we hasten to point out that the bacteria present may well be commensal populations that have no adverse consequences. We also caution that using antibiotics in semen before assisted reproductive technology treatment may damage sperm and even the quality of the embryo. However, as Dr. Baka points out, many questions remain unanswered. Perhaps none is more intriguing than whether bacterial populations are being shared between heterosexual partners and how this might affect the risk to disease in males and females. Larry J. Forney, Ph.D.a Chen Xu, M.D., Ph.D.b Dongsheng Hou, M.D., Ph.D.c a Institute for Bioinformatics and Evolutionary Studies, University of Idaho, Moscow, Idaho; b Department of Histology and Embryology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghei, People's Republic of China; and c Shandong Provincial Hospital, Shandong University, Shandong, People's Republic of China January 22, 2014 http://dx.doi.org/10.1016/j.fertnstert.2014.01.043

REFERENCE 1.

Hou D, Zhou X, Zhong X, Settles M, Herring J, Wang L, et al. Microbiota of the seminal fluid from healthy and infertile men. Fertil Steril 2013;100:1261–9.

VOL. 101 NO. 4 / APRIL 2014