Larry I. Lipshultz

APOPTOTIC FREQUENCY IS INCREASED IN SPERMATOGENIC MATURATION ARREST AND HYPOSPERMATOGENIC STATES

PURPOSEnIncreased testicular apoptosis has been observed in maturation arrest and hyposper-matogenesis states in rodent models, but this process has not yet been characterized in humans. We hypothesized that increased cell death present with accelerated apoptosis is significant in pathophysiology of many male infertility states associated with abnormal spermatogenesis. We examined frequency of apoptotic bodies in human testis biopsy specimens from infertile men using morphometric analysis of hematoxylin and eosin stained paraffin sections.nnnMATERIALS AND METHODSnTestis biopsy specimens were obtained for routine clinical purposes from azoospermic and severely oligozoospermic men and were stained with hematoxylin and eosin. Apoptotic bodies were identified using established morphometric criteria. Apoptotic indexes, defined as apoptotic bodies per total number of cells and per Sertoli cells, were calculated after counting all intratubular spermatogenic cells and Sertoli cells in 20 tubules.nnnRESULTSnA total of 51 biopsies was performed in 50 men. Significantly increased apoptotic body per total cell and apoptotic body per Sertoli cell ratios were observed in maturation arrest and hypospermatogenesis states in comparison to Sertoli cell only and normal spermatogenesis (p < 0.05, Mann-Whitney test).nnnCONCLUSIONSnIncreased apoptosis in maturation arrest and hypospermatogenesis states compared to normal but obstructed spermatogenesis and Sertoli cell only were observed, indicating a prominent role for this form of programmed cell death in human male infertility.

BILATERAL NERVE GRAFT DURING RADICAL RETROPUBIC PROSTATECTOMY: 1-YEAR FOLLOWUP

PURPOSEnWith the interposition of a sural nerve graft to replace resected cavernous nerves at radical retropubic prostatectomy, we have previously reported the return of effective erectile function. We determine the efficacy of this procedure in a series of men with at least 1-year followup.nnnMATERIALS AND METHODSnA total of 12 potent men (mean age plus or minus standard deviation 57 +/- 6 years) with clinically localized prostate cancer underwent radical retropubic prostatectomy, with deliberate wide bilateral neurovascular bundle resection and placement of bilateral nerve grafts. A series of patient and partner erectile dysfunction questionnaires, and patient interviews were performed at 3, 6, 12 and 18 months postoperatively. Only results for those men with a followup of 12 months or greater (mean 16 +/- 4) are presented. A control group of 12 men who had undergone bilateral nerve resection but declined nerve graft placement, was also followed.nnnRESULTSnOf the 12 men 4 (33%) had spontaneous medically unassisted erections sufficient for sexual intercourse with vaginal penetration. An additional 5 (42%) men describe 40 to 60% spontaneous erections, with fullness, no rigidity and not able to penetrate. Overall, 9 (75%) men had return of erectile activity. No demonstrable erections occurred before 5 months postoperatively. The greatest return of function was observed at 14 to 18 months after surgery.nnnCONCLUSIONSnThis surgical technique has minimal morbidity and represents a significant advance in prostate cancer surgery in men requiring bilateral nerve resection. Our study clearly demonstrates recovery of erectile function in men who underwent bilateral nerve graft placement during radical retropubic prostatectomy when both cavernous nerves were deliberately resected.

In vitro fertilization in couples with male factor infertility

The data from 83 consecutive in vitro fertilization-embryo transfer cycles were examined with emphasis on the presence of subfertile male parameters, including abnormal sperm density, motility, morphologic features, or an abnormal result in the sperm penetration assay (zona-free hamster ova penetration). There were 25xa0cycles (21 couples) in which there was a solitary or coexistent abnormal male factor. The presence of a male factor was associated with a lower fertilization rate and fewer embryos transferred. Statistically important reductions in the fertilization rate occurred with reduced sperm density and abnormal motility, but not with abnormal morphologic features. The parameter that was most significantly associated with human ova fertilizability was the ability to exceed two penetrations per egg in the sperm penetration assay.

Testis biopsy image analysis effectively quantifies spermatogenic cell types.

PURPOSEnEvaluation of the testis biopsy specimen in male infertility has been hampered by the qualitative rather than quantitative nature of routine histological techniques and interpretation. Although useful, flow cytometry has demonstrated significant limitations due to its inability to differentiate among haploid cell types, spermatozoa and spermatids. Thus, deoxyribonucleic acid image analysis was performed on testis biopsy specimens to combine the advantages of quantitative ploidy and morphological characteristics to develop a more effective quantitative system.nnnMATERIALS AND METHODSnImage analysis using a specifically designed filter was performed on Feulgen stained 5 microns. sections of paraffin embedded testicular tissue. The archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia and normal spermatogenesis on routine hematoxylin and eosin processed tissue.nnnRESULTSnThe overall findings (mean plus or minus standard deviation) in 20 biopsies from 18 men are haploid 54.2 +/- 5.6, diploid 31.2 +/- 5.2 and tetraploid 15.0 +/- 4.6%. Spermatozoa and spermatids contributed 23.2 +/- 5.7 and 30.9 +/- 7.6% to the total cell content, respectively. A mean of 2,462 +/- 254 cells were counted per case.nnnCONCLUSIONSnWe used image analysis to evaluate testicular biopsies. This technique can quantitatively determine the percentages of various cell types within the seminiferous tubules. Furthermore, it can differentiate between spermatozoa and spermatids based on morphological characteristics. This technique, which combines ploidy and cell morphology characteristics, represents a significant advancement in the interpretation of the testicular biopsy, and has implications for redefining the presently used system of qualitative interpretative assessment.

Testicular Touch Preparation Cytology

PURPOSEnThe applications, technique and limitations of testicular touch preparation cytology in evaluation of the infertile man are described.nnnMATERIALS AND METHODSnThe technique and histological results of testicular touch preparation cytology for normal specimens and various pathological conditions of male infertility are described in detail.nnnRESULTSnTesticular touch preparation cytology is able to differentiate between late spermatids and mature spermatozoa, as well as to identify other spermatogenic elements.nnnCONCLUSIONSnTouch preparation cytology has 2 important purposes: 1) distinguishing between maturation arrest at the late spermatid stage and normal spermatogenesis, and 2) determining whether obstruction is the cause of azoospermia. While testis biopsy is the gold standard, touch preparation cytology is an extremely important adjunct because of its ability to define whole sperm. The urologist and pathologist are better able to make a diagnosis when routine biopsy and touch preparation cytology are performed.

IMAGE ANALYSIS ASSESSMENT OF THE ABNORMAL TESTIS BIOPSY IN MALE INFERTILITY

PURPOSEnWe previously demonstrated that testis biopsy image analysis is an effective method for quantifying intratubular spermatogenic cells in the obstructed testis with normal spermatogenesis. As an extension of the initial report, we describe using the quantitative ploidy and morphological characteristics of cells counted with image analysis in abnormal testis biopsies obtained for a male infertility evaluation.nnnMATERIALS AND METHODSnImage analysis using a specifically designed filter was performed on Feulgen stained 5 microns, sections of paraffin embedded testicular tissue. Archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia or severe oligospermia. Qualitative classification was based on standard evaluation of hematoxylin and eosin processed tissue.nnnRESULTSnThere were 62 biopsies performed in 58 men. Significant differences in the intratubular content of haploid (spermatozoa and spermatids), diploid and tetraploid cells were found among the 5 categories of abnormalities: the Sertoli-cell-only syndrome, spermatocyte arrest, spermatid arrest, hypospermatogenesis and normal spermatogenesis. Moderate variability was found in the proportion of cell types in spermatid arrest and hypospermatogenesis.nnnCONCLUSIONSnTestis biopsy image analysis provides a quantitative method for categorizing abnormalities of intratubular cell content present in male infertility states by using deoxyribonucleic acid content and morphology characteristics. The limitations of the present qualitative analysis system are emphasized by the moderate variability evident within the current categories of spermatid arrest and hypo-spermatogenesis states.

IMAGE ANALYSIS ASSESSMENT OF TESTICULAR TOUCH PREPARATION CYTOLOGIES EFFECTIVELY QUANTIFIES HUMAN SPERMATOGENESIS

AbstractPurposes: We have recently demonstrated that computer assisted image analysis of paraffin embedded testicular tissue based on deoxyribonucleic acid content and morphology characteristics is an effective method for the quantitative assessment of spermatogenesis. We assess the use of testicular touch preparation image analysis as a technique for quantification of spermatogenesis.Materials and Methods: Air dried, touch imprints of testicular tissue from obstructed azoospermic and severely oligozoospermic patients were obtained at the time of biopsy. Image analysis using a filter based on deoxyribonucleic acid content and cellular morphological characteristics was performed on Feulgen stained touch preparation imprints as well as paraffin embedded sections.Results: Image analysis of 52 testicular touch preparations from 48 azoospermic or severely oligozoospermic men revealed significant differences (p <0.05) in the percentages of spermatid and spermatozoa, and 2N and 4N cells among seminiferous tubule...

Correlation between simultaneous PSA and serum testosterone concentrations among eugonadal, untreated hypogonadal and hypogonadal men receiving testosterone replacement therapy

The primary objective of this study was to correlate simultaneous measures of prostate-specific antigen (PSA) and serum testosterone among large samples of eugonadal, untreated hypogonadal and hypogonadal men treated with testosterone replacement therapy (TRT). From 2001 to 2007, laboratory records were reviewed to identify men who underwent simultaneous measurement of PSA and serum testosterone levels. The data were stratified based on three groups of men: group 1 consisted of eugonadal men (T>300u2009ng per 100u2009ml) evaluated for BPH, reproductive failure or sexual dysfunction; group 2 consisted of untreated hypogonadal men (T<300u2009ng per 100u2009ml); and group 3 comprised symptomatic hypogonadal men receiving TRT. Correlations were found between PSA (total and free fractions) and total serum testosterone levels among the three groups. Group 1: eugonadal men (n=385 patients), mean PSA and serum testosterone were 1.60u2009ngu2009ml−1 and 484u2009ngu2009100u2009ml, respectively. There was no significant correlation between PSA and total serum testosterone levels (r=−0.01, P=0.8). Group 2: untreated hypogonadal men (n=229 patients), mean PSA and serum testosterone were 1.49u2009ngu2009ml−1 and 269u2009ng per 100u2009ml, respectively. There was no significant correlation between PSA and total serum testosterone levels (r=0.03, P=0.6). Group 3: hypogonadal men on TRT (n=229 patients and 994 individual samples analyzed) mean PSA and serum testosterone were 1.50u2009ngu2009ml−1 and 555u2009ng per 100u2009ml, respectively. There was no significant correlation between PSA and serum testosterone levels (r=−0.005, P=0.9). Mean total serum testosterone levels were increased significantly (P<0.001) following treatment. Mean PSA levels did not increase in a statistically or clinically significant manner following TRT (mean PSA increase from baseline 0.05u2009ngu2009ml−1, P=0.6). In conclusion, TRT does not appear to significantly influence serum PSA expression and no significant correlation was identified between PSA and serum testosterone among eugonadal, untreated hypogonadal and hypogonadal men receiving TRT.

Evaluation of the Infertile Male

With recently evolved diagnostic and therapeutic techniques now available for the infertile couple, even the most severe male factor problems are potentially treatable. Men previously considered irreversibly infertile may now initiate their own biologic pregnancies. Many of these significant changes have been provided by intracytoplasmic sperm injection (ICSI), one of the major medical milestones of the 20th century. Despite this important technical advance, evaluation of the male patient remains essential, because a male factor is found to be contributory in up to 50% of infertile couples. The temptation to omit or to offer a compromised evaluation of men, based on the erroneous belief that the success of ICSI supersedes the need for a proper andrologic assessment, must be resisted. Of patients with male factor infertility, 70% can be treated without ICSI. Of azoospermic men, 50% can become candidates for ICSI.

Cancer risk among children born after assisted conception.

From the Institute of Child Health, University College London (C.L.W., B.J.B., A.G.S.), and the Reproductive Medicine Unit, University College London Hospitals (M.D.), London; the Childhood Cancer Research Group, Department of Paediatrics, University of Oxford, Oxford (K.J.B., C.A.S., M.F.G.M.); and the Paediatric Oncology Department, Royal Hospital for Sick Children, University of Edinburgh, Edinburgh (W.H.W.) — all in the United Kingdom. Address reprint requests to Dr. Sutcliffe at the General and Adolescent Paediatric Unit, Institute of Child Health, University College London, 30 Guilford St., London WC1N 1EH, United Kingdom, or at a.sutcliffe@ucl.ac.uk.n engl j med 370;10 nejm.org march 6, 2014 974 mutations in 192 Chinese patients with primary ovarian insufficiency did not identify a mutation in DMC1.4 We suggest that mutations in genes encoding proteins that regulate meiosis can result in autosomal recessive primary ovarian insufficiency in humans, although our findings await confirmation by independent groups. However, our results are consistent with the finding that Hfm1-deficient mice are infertile.5 Further investigation of HFM1 in larger series of women with primary ovarian insufficiency and in infertile men is warranted.