going 40 cycles of IVF/ICSI were evaluated. 15 cycles of ICSI utilized freshand 25 cycles frozen-thawed sperm/germ cells. In the control group, 18patients with acquired and 4 patients with congenital OA resulted in 12 freshand 28 frozen-thawed ICSI cycles. IVM was initially performed for 24, 48or 72 hours prior to ICSI, and then routinely 48 hours after 2000. Sperm/testicular tissue was incubated in protein supplemented HTF at 37o C in 5%CO2 both with and without LH/FSH. The presence of viable germ cells,motility, morphology, fertilization rate, cleavage rate, embryo developmentand pregnancy outcomes were compared among groups. Chi-squared anal-ysis, Student’s t-test for paired groups and ANOVA were used as appro-priate with p �0.05 considered significant.

Results: The patients age ranged from 29–56 yr. NOA diagnoses in-cluded hypospermatogenesis (n � 7), maturation arrest (n � 6), priorchemo/radiation exposure (n � 4), endocrinopathy (n � 3), and refractoryanejaculation (n � 7). OA diagnoses included congenital absence of the vas(n � 4) and prior vasectomy (n � 18). Compared with OA, patients withNOA had significantly decreased sperm viability, motility, and normalmorphology in both fresh and frozen-thawed samples at ICSI. However thefertilization rate in fresh and frozen thawed NOA cycles of 71.6% (126/176)and 64.5% (187/290) did not differ from OA cycles (72.6 and 70.4%) (p�0.05) nor did cleavage, embryo quality, viable embryos for transfer, orpregnancy rates (35.7 and 33.3% vs. 27.3 and 46.2%, (p �0.05). IVM offresh and frozen-thawed sperm/germ cells for greater than 24 hours in NOAresulted in a significant increase in motile/viable germ cells for ICSI (5.8 vs.19.5% and 6.3 vs. 23.1%, p �0.05) and fertilization (50.4 vs. 70.2% and48.7 vs. 61.1%, p � 0.05), in both fresh and frozen-thawed cycles, respec-tively.

Conclusions: Although sperm/germ cell quality following TESE for NOAis significantly impaired when compared with OA, IVM of both fresh andfrozen-thawed specimens significantly improves clinical ICSI success. Sim-ilar to previous experience with OA, IVM for more than 24 but less than 72hours results in improved recovery of viable gametes for use in ICSI andresulting fertilization. Although immature spermatozoa may benefit fromgonadotropin stimulation at IVM, perhaps via effects on responsive testic-ular cells in culture, we found such treatment to be of no clear benefit in themajority of NOA diagnoses.

Supported by: None.

Tuesday, October 15, 20024:15 P.M.

O-164

Fluorescence “in situ” hybridization (FISH) analysis of chromosomalabnormalities in testicular and epididymal spermatozoa. Lorena Ro-drigo, Carmen Rubio, Carlos Simon, Jose A. Remohi, Antonio Pellicer,Manuel Gil-Salom. Inst Valenciano de Infertilidad, Valencia, Spain; InstValenciano de Infertilidad;Department of Pediatric, Obstetrics and Gyne-cology, Univ of Valencia, Valencia, Spain; Inst Valenciano de Infertilidad;Department of Pediatric, Obstetrics and Gynecology, Univ of Valencia,Valencia, Spain; Inst Valenciano de Infertilidad;Department of Surg, Univof Valencia, Valencia, Spain.

Objective: An increased incidence of numerical chromosomal abnormal-ities in ejaculated spermatozoa has been reported in infertile patients whencompared to fertile donors. However, data on the incidence of aneuploidy insurgically retrieved spermatozoa from testicle or epididymis are still limitedand controversial. The objective of the present study is to analyze the ratesof numerical chromosomal abnormalities in testicular and epididymal sper-matozoa obtained from azoospermic patients who are candidates for intra-cytoplasmic sperm injection (ICSI).

Design: A prospective controlled study including patients with obstruc-tive (OA) and non-obstructive azoospermia (NOA).

Materials/Methods: A total of 19 samples from azoospermic patients withnormal blood karyotypes were analyzed: two OA patients underwent epi-didymal sperm aspiration for further ICSI attempts; in 7 OA patients and 10NOA patients testicular sperm extraction (TESE) was performed for thesame purpose. Ejaculated spermatozoa from 5 normozoospermic fertiledonors were also evaluated as control group. Sperm samples were fixed afterhypotonic treatment. Sperm nuclei were decondensed and five chromo-somes were evaluated by FISH on different slides. Triple FISH was per-formed for chromosomes X, Y and 18, and dual FISH for chromosomes 13and 21 (Vysis Inc. Downers Grove, IL, USA). FISH results in tailed

spermatozoa were compared among the three groups and with the controlgroup. Individual FISH results were also compared with the control group,and were considered as abnormal when a statistically significant increase inany of the analysed parameters was observed. Statistical analysis wasperformed using Chi square-test with Yates correction when indicated.

Results: Epididymal sperm results were comparable to the control group.Testicular spermatozoa from OA patients showed a significant increase insex chromosome disomy rate. Testicular sperm from NOA patients pre-sented significant increases in disomy rates for all of the chromosomesevaluated and in the diploidy rate (Table). Individual FISH results wereabnormal in 2 testicular OA samples (29%) and in 6 testicular NOA samples(60%). Both epididymal sperm samples were normal.

Epididymal(OA)

(n � 2)

Testicular(OA)

(n � 7)

Testicular(NOA)

(n � 10)Control(n � 5)

No. sperm scored X/Y/18 4,204 4,543 2,048 50,404% Sex chr. Disomy 0.19 0.46a,b 1.17a,b,c 0.24% Disomy 18 0.07 0.00 0.15a,c 0.03No. sperm scored 13/21 4,135 2,736 2,262 50,373% Disomy 13 0.12 0.15 0.31a 0.10% Disomy 21 0.07 0.26 0.49a,b 0.14Total no. sperm scored 8,339 7,279 4,310 100,777% Diploidy 0.16 0.12 0.37a,b,c 0.11

a P � 0.05 vs. control, b P � 0.05 vs. epididymal, c P � 0.05 vs. testicular(OA)

Conclusions: Testicular spermatozoa from azoospermic patients under-going TESE for ICSI show an increased incidence of chromosomal abnor-malities, mainly for sex chromosomes, being paticularly high in patientswith non-obstructive azoospermia.

Supported by: IMPIVA.

Tuesday, October 15, 20024:30 P.M.

O-165

False-positive Y chromosome microdeletion in a fertile male. Alan R.Thornhill, Adam J. Guenther, Gina M. Barbarotto, Karen Snow. MayoClin, Rochester, MN.

Objective: The pathogenic relationship between the presence of Y chro-mosomal microdeletions and male infertility is unclear. Nevertheless acausal relationship is thought to be probable when loci deleted in infertilemales are present in fertile males. PCR analysis of the Y chromosome isnow routinely performed in the evaluation of the infertile male although,until recently, there has been no consensus on how the diagnosis should beperformed and which loci or markers should be analyzed. The EuropeanAcademy of Andrology (EAA) published guidelines for the moleculardiagnosis of Y chromosomal microdeletions in 1999. Following theseguidelines, our laboratory developed assays that incorporated the suggestedprimer pairs for the recommended Sequence Tagged Sites (STS). Theobjective of this study was to assess the sensitivity and specificity of our Ychromosome microdeletion assay by testing DNA from fertile men andblinded reference samples. Our clinical assay requires follow up of anysample apparently deleted for one or more of the STS markers.

Design: Prospective study.Materials/Methods: A number of fertile samples collected at Mayo Clinic

(n�101), and blinded reference samples (n�20) sent from outside labora-tories (including an external quality assessment scheme organized by theEAA) were tested in our laboratory as part of the validation to provide aclinical assay. Two multiplex PCR assays were optimized, each of whichexamined STS markers in the center of the Azoospermic factor (AZF) a, band c regions of the Y chromosome.

Results: We correctly identified all but one of the 121 samples (accordingto the expected result based on fertility or previous testing at anotherlaboratory). Of the 20 blinded reference samples, seven had known dele-tions in AZFa, AZFb, or AZFc. Using our assay, all reference samples werediagnosed correctly to give an analytical sensitivity of 100%. Of the 101

FERTILITY & STERILITY� S63