Pilot study of the correlation of multiphoton tomography of ex vivo human testis with histology.
OBJECTIVE
Although microdissection testicular sperm extraction has become first line therapy for sperm retrieval in men with nonobstructive azoospermia, there are challenges to the procedure, including difficulty differentiating between seminiferous tubules with normal and abnormal spermatogenesis. Multiphoton microscopy illuminates tissue with a near infrared laser to elicit autofluorescence, which enables real-time imaging of unprocessed tissue without labels. We hypothesized that we could accurately characterize seminiferous tubular histology in humans using multiphoton microscopy.


METHODS
Seven men with normal or abnormal spermatogenesis underwent testicular biopsies, which were imaged by multiphoton microscopy. We assessed these images in blinded fashion. The diagnosis rendered with multiphoton microscopy was then correlated with that of hematoxylin and eosin stained tissue. We evaluated the ability of multiphoton microscopy to differentiate normal from abnormal seminiferous tubules by examining autofluorescence characteristics and diameters, as imaged by multiphoton microscopy. Assessment was repeated with stained slides and results were compared.


RESULTS
The overall concordance rate between multiphoton microscopy and stained slides was 86%. The seminiferous tubules of patients with nonobstructive azoospermia were smaller than those of controls when measured by multiphoton microscopy and staining (p <0.05). The proportion of normal tubules and the diameters obtained with multiphoton microscopy were not different from those obtained with hematoxylin and eosin (p >0.05).


CONCLUSIONS
Multiphoton microscopy can be used to differentiate normal from abnormal spermatogenesis. Its characterization of seminiferous tubular architecture is similar to that provided by hematoxylin and eosin staining. Further investigation of the clinical applications of multiphoton microscopy may improve surgical sperm retrieval outcomes for patients with nonobstructive azoospermia.
Department of Urology, Weill Cornell Medical College, New York, New York, USA.
Bobby B
BB
Najari
Ranjith
R
Ramasamy
Joshua
J
Sterling
Amit
A
Aggarwal
Seema
S
Sheth
Philip S
PS
Li
Justin M
JM
Dubin
Sagit
S
Goldenberg
Manu
M
Jain
Brian D
BD
Robinson
Maria
M
Shevchuk
Douglas S
DS
Scherr
Marc
M
Goldstein
Sushmita
S
Mukherjee
Peter N
PN
Schlegel
D000328:Adult; D053713:Azoospermia / Q000473:pathology* / Q000628:therapy; D001706:Biopsy / Q000379:methods; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D008297:Male; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008866:Microsurgery / Q000379:methods*; D008875:Middle Aged; D012016:Reference Values; D012671:Seminiferous Tubules / Q000473:pathology; D012680:Sensitivity and Specificity; D012708:Sertoli Cells / Q000473:pathology; D053625:Sperm Retrieval; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000473:pathology*