fibroblast feeder layer and incubated with embryonic stem cell (ES) medium O-209 Wednesday, October 27,

ctober 27, 2010 04:15 PM

and basic fibroblast growth factor. After 16 days, a small number of colonies
were visible by microscopy. SEMEN CHARACTERISTICS OF A LARGE COHORT SHOW
RESULTS: We were able to collect ES-like cells derived from infertile PROTAMINE 1 (P1) TO PROTAMINE 2 (P2) RATIO IS INDEPEN-
men. Short tandem repeat analysis confirmed that these colonies were in- DENT OF OTHER SPERM PARAMETERS. L. Nanassy, D. Carrell. An-
deed derived from testis tissue. The normal testis cells expressed OCT4, drology and IVF Laboratories, Department of Surgery, University of Utah
NANOG, STELLA, VASA, and DAZL. The colonies expressed OCT4, School of Medicine, Salt Lake City, UT; Department of Obstetrics and
NANOG, and STELLA, but did not express the germ cell markers Gynecology, University of Utah School of Medicine, Salt Lake City, UT;
VASA and DAZL. We examined embryoid body formation and teratoma Department of Physiology, University of Utah School of Medicine, Salt
formation to assess pluripotency both in vitro and in vivo. It was not pos- Lake City, UT.
sible to generate complete iPS cells from all infertile patients, even using
OBJECTIVE: Although, abnormal protamination has been associated
identical methods. Some iPS cells could not be cultured after we collected
with male infertility, prior studies have suffered from small sample size
ES-like cells from the first step.
and therefore have not suitably addressed the question of the frequency of ab-
CONCLUSION: Although we were unable to generate iPS cells from all
normal P1/P2 ratio in normozoospermic men. The aim of the study was to
infertile patients, development of a more efficient and straightforward
evaluate the relationship between sperm parameters and P1/P2 ratio in a large
method of generating iPS cells remains our goal. Analysis of iPS cells
number of subjects.
from infertile men may help to identify the mechanisms responsible for
DESIGN: Retrospective analysis.
male infertility.
MATERIALS AND METHODS: Six hundred and thirty-eight subjects
were included in the study. Semen analysis was carried out according to
WHO (2000) standards. The P1/P2 ratio was determined using acetic acid-
urea gel electrophoresis. Differences between groups were determined by
O-208 Wednesday, October 27, 2010 04:00 PM X2 analysis and student t-test (p<0.05 was considered significant).
MUTATIONS IN THE MSH5 MISMATCH REPAIR GENE IN NON- RESULTS: Four hundred and two subjects had abnormal semen parame-
OBSTRUCTIVE AZOOSPERMIC MEN (NOA). S. Mukherjee, ters (< 20 M/ml sperm, and/or < 40% progressive motility, and/or < 30%
J. W. Weedin, J. B. Addai, L. I. Lipshultz, D. J. Lamb. Department of Urol- normal head morphology), and 236 subjects were classified as normozoo-
ogy, Baylor College of Medicine, Houston, TX; Department of Molecular spermic. The P1/P2 ratio was statistically different (p<0.05) between sub-
and Cell Biology, Baylor College of Medicine, Houston, TX. jects with normal (1.13  0.0342) and abnormal (1.24  0.0285) semen
parameters. We previously established a normal reference range of 0.53 to
OBJECTIVE: Defects in mismatch repair (MMR) genes are associated 1.43 for P1/P2 ratio based on fertile, normozoospermic population. We found
with genomic instability and development of cancer. MMR mutation carriers a significantly higher frequency (p¼0.0090) of subjects in the normal P1/P2
are also at high risk for extracolonic malignancies as occurs in Lynch syn- range and a lower frequency (p¼0.0090) of subjects in the high P1/P2 range
drome. Interestingly, MMR (Pms2, Mlh3 and Mlh1)-deficient mice display in the normozoospermic group (87.71% [207/236] versus 79.60 [320/402];
infertility associated with abnormal chromosome pairing in meiosis. 10.59% [25/236] versus 18.41% [75/402], respectively). Sperm concentra-
MSH5 is a MutS homolog that plays an important functional role in meiotic tion was significantly reduced in the high P1/P2 group (80.67  5.967 M/
recombination during Holliday Junction (HoJo) formation. MSH4 interacts ml) compared to control group (104.54  3.265 M/ml; p<0.05). Sperm mo-
with MSH5 forming a heterodimer that uniquely binds to the HoJo crossover tility and normal head morphology did not differ between low, normal and
region, suggesting a role in both recombination and crossover interference. high P1/P2 groups (p>0.05).
Our data shows evidence of MSH5 deficiencies in men with non-obstructive CONCLUSION: Surprisingly, and contrary to small, early reports, P1/P2
azoospermia. ratio seems to be independent of sperm count, motility and morphology. It is
DESIGN: Cohort of 371 infertile men and 66 fertile controls were tested likely that aberrant protamination is indicative of other abnormalities during
for mutations in the MSH5 gene. spermiogenesis, and independent of spermatogenesis.
MATERIALS AND METHODS: Mutation analysis of the 25 exons of
MSH5 gene spanning 22856 bp was performed on genomic DNA. In partic-
ular, exon 2 was amplified as it encompasses the interaction domain of MSH4 O-210 Wednesday, October 27, 2010 04:30 PM
and MSH5. The nuclear localization signal (KKRKR, 406-409 aa) mapped to
INCREASED SPERM DNA DAMAGE IS ASSOCIATED WITH PROT-
exon 15 was also evaluated.
AMINE PACKAGING ANOMALIES. M. M. Peart, K. R. Chohan. Depart-
RESULTS: Genomic DNA analyses of exon 2 of MSH5 gene by denatured
ment of Pathology, SUNY Upstate Medical University, Syracuse, NY.
high performance chromatograghy (DHPLC) revealed polymorphisms and
genetic abberations. Sequencing data revealed a variant (C84A) that altered OBJECTIVE: To determine the relationship between sperm DNA damage
codon 29 of the MSH5 gene, resulting in a proline to leucine change (P29L) and protamination relative to semen parameters.
in 7 infertile men and none of the 66 fertile controls. All 7 infertile men had DESIGN: Prospective study.
a sperm density of less than 1  106/ml. MATERIALS AND METHODS: Semen samples from 126 non smoking
CONCLUSION: The P29L mutation located in the interacting domain patients were evaluated according to WHO 3rd criteria. Based on semen
may lead to weakened protein interaction with MSH4, thus affecting genetic quality, patients were allocated to normozoospermic (N), teratozoospermic
stability and DNA recombination. Consistent with the structural role as a-he- (T), oligoteratozoospermic (OT), asthenoteratozoospermic (TA) and oligoas-
lix breaker, the mutation of proline to leucine may disrupt the secondary thenoteratozoospermic (OAT) groups. Sperm viability and hypo-osmotic
structure of the protein, resulting in defective interaction with MSH4. In vitro swelling tests were also performed. Chromomycin A3 (CMA3) staining
interaction studies will define the functional consequences of this mutation in was used to assess protamine content and DNA damage was evaluated by
meiosis. TUNEL assay. Data were analyzed with Pearson correlation and ANOVA
Supported by: PO1HD36289 to DJL from the NIH. while Dunn or Holm-Sidak methods were used for multiple comparisons.

Semen Paramters in Various Groups

Age Volume Total Motility Progressive Count Normal

Groups (yrs) (ml) (%) Motility (%) (mill/ml) Morphology DNA damage (%)

N n¼44 29.7  8.1a 2.9  1.1 65.7  8.2a 58.3  9.6a 164.4  88.6a 36.1  6.7a 10.9  5.8a
T n¼54 32.9  7.5a,b 3.5  1.7 62.1  7.3a 51.6  9.3b 113.3  63.5a 17.8  6.4b 14.5  9.1a
OAT n¼8 37.8  7.2b 3.0  2.5 34.2  12.6b 20.0  10.8c 10.4  6.1b 9.0  5.4c 36.4  13.8b
TA n¼13 37.6  8.6b 2.1  1.0 33.1  11.3b 23.2  13.3c 115.6  99.9a 13.4  7.8c 23.1  8.1b
OT n¼7 34.0  10.3a,b 4.4  2.9 61.9  6.2a 44.4  8.1b 10.2  5.9b 13.1  9.0c 8.3  4.3a

Values are Mean  SD and different superscripts in columns indicate significant differences among groups (P<0.05).

FERTILITY & STERILITYÒ S61

 RESULTS: A significant correlation between CMA3 positive cells and MATERIALS AND METHODS: 20 pubescent male Wistar Hannover rats
DNA damage was observed when damage was greater than 15% in the were fed deionized water containing 0 or 100 mg/L Cd ad libitum for 1 or 8
OAT and T groups. A negative correlation was found between CMA3 positive weeks. Whole testis RNA was template for cRNA synthesis. Biotinylated
cells and sperm morphology. cRNAs were hybridized to Affymetrix Rat 230.2 microarrays, and data-
CONCLUSION: Decreased protamination positively correlates with DNA mined by GeneSpring GX 7.3.1. Five microarrays per time/dose combination
damage that exceeds 15% in OAT and T patients. Sperm DNA damage in- condition (one per rat) were statistically analyzed.
creases with deteriorating semen parameters. Assessment of sperm DNA RESULTS: Actin-related genes studied fell into two groups: [1] expression
damage, especially in oligoasthenoteratozoospermic and asthenoteratozoo- independent of Cd but changed with time, and [2] expression down-regulated
spermic patients, may be beneficial in assisted reproduction. by Cd at 1 week or 8 weeks. At 1 week, Cd-regulated genes largely controlled
shape/morphogenesis, and kinases/phosphatases. At 8 weeks, Cd-modulated
genes controlled actin filaments (interacting proteins; polymerization/depo-
lymerization), cell projection formation and cell motility/migration.
O-211 Wednesday, October 27, 2010 04:45 PM CONCLUSION: Cd differentially affected adolescent vs. adult testes. Our
QUANTITATIVE SHOTGUN PROTEOMIC ANALYSIS OF SEMI- data are consistent with Cd-effected actin gene expression disruption contrib-
NAL PLASMA FROM MEN WITH SPINAL CORD INJURY-IN- uting to apoptosis and decreased spermatogenesis in rat testes. Studies of
DUCED ANEJACULATION. B. F. da Silva, C. R. Ferreira, gene expression in human testes biopsies should determine relevance to
M. N. Eberlin, J. S. Garcia, G. H. M. F. Souza, R. P. Bertolla. Division of varicocele-associated infertility.
Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Supported by: PHS Grant ES10496 (SHB); FIMR Faculty Research Award
Paulo, SP, Brazil; ThoMSon Mass Spectrometry Laboratory, Institute of (SHB).
Chemistry, University of Campinas, Campinas, SP, Brazil; Mass Spectrom-
etry Applications Research and Development Laboratory, Waters Corpora- SOCIETY FOR REPRODUCTIVE ENDOCRINOLOGY AND
tion, Alphaville, SP, Brazil; Department of Exact Sciences, Alfenas INFERTILITY
Federal University, Alfenas, MG, Brazil.
O-213 Wednesday, October 27, 2010 03:45 PM
OBJECTIVE: Most spinal cord injured (SCI) patients present a semen pro-
file mainly characterized by normal sperm count and abnormal sperm motility. RACIAL/ETHNIC DIFFERENCES IN OVARIAN RESERVE
A possible correlation between this problem and abnormal semen constituents MARKERS. M. P. Rosen, E. B. Johnstone, C. Addauan-Andersen,
seems to exist. To further explore this information and to potentially identify B. Sternfeld, C. McCulloch, M. I. Cedars. Obstetrics, Gynecology, and Re-
proteins involved in SCI infertility, a quantitative shotgun proteomics strategy productive Sciences, University of California San Francisco, San Francisco,
was employed to observe the seminal plasma proteome in these men. CA; Division of Research, Kaiser Permanente, Oakland, CA; Epidemiology
DESIGN: Prospective study. & Biostatistics, University of California San Francisco, San Francisco, CA.
MATERIALS AND METHODS: Semen was obtained from men with
SCI. Electroejaculation (EEJ) was performed in 6 patients, penile vibratory OBJECTIVE: Historical evidence suggests that African Americans and
stimulation (PVS) in 6 other. 10 patients without SCI presenting normal se- Hispanics have earlier ages of menopause than Caucasians, and Asians, later.
men were included as controls. Samples were pooled by group and submitted We utilize a large, population-based cohort to evaluate racial/ethnic differ-
to nanoUPLC tandem nanoESI-MSE quantitative shotgun proteomics. Sta- ences in markers of ovarian age.
tistically identified proteins were analyzed for interactome networks using DESIGN: Cross-sectional.
Cytoscape and classified according to Gene Ontology. MATERIALS AND METHODS: Women aged 25-45 with ovulatory men-
RESULTS: A total of 881 proteins were identified, 325 differentially ex- ses at 22-35 day intervals, enrolled in the population-based Ovarian Aging
pressed between the three groups – 98 exclusively in the EEJ group, 135 in study, underwent transvaginal ultrasound for antral follicle count (AFC)
the PVS group, and 40 in controls. Moreover, 31 were overexpressed in men and blood testing for antimullerian hormone (AMH) between cycle day 2
with EEJ, 15 in PVS, and 6 in controls. Cytoscape analysis demonstrated 8 inter- and 4. Generalized linear models were used to assess for ethnic differences
actome clusters, of which one was present only in men with PVS, responsible for in AMH and AFC, adjusted for age.
response to hypoxia. A main cluster responsible for reproduction and ion binding RESULTS: This study includes 694 subjects: Caucasians (n¼244), Afri-
was observed in all groups, but with 6 different proteins in men with EEJ and 4 in can American (n¼175), Chinese (n¼131), and Latinas(n¼144). In Table 1
men with PVS, demonstrating putative pathways for altered semen quality. are the means and SDs for AMH and antral follicle count (AFC), stratified
CONCLUSION: Men presenting with anejaculation due to SCI present al- by age and ethnicity. AMH and AFC decline with age. Based on the regres-
teration in the expression of a number of proteins when compared to controls sion analysis, AMH and AFC both differ by ethnicity: Caucasian women
with normal semen. The use of a quantitative shotgun proteomics approach have higher levels of AMH compared to Latina women (p ¼ 0.006) while
has increased sensitivity and the dynamic range for protein identification. AFC is significantly higher in African-Americans than Chinese (p ¼ 0.03).
The information from this study may allow us to (i) better understand the
pathways leading to normal and/or altered semen quality and (b) determine
biomarkers for therapeutic intervention.
Supported by: CNPq. Mean  SDs for AMH and AFC by Age

Age Caucasian Latina AA Chinese

O-212 Wednesday, October 27, 2010 05:00 PM
25-30 AMH 42  19 40  18 35  8 37  27
CADMIUM ALTERS TESTICULAR EXPRESSION OF GENES REG- AFC 22  10 23  9 23  11 18  8
ULATING THE STRUCTURE AND FUNCTION OF THE ACTIN CY- 31-35 AMH 36  24 25  17 28  6 36  28
TOSKELETON. S. H. Benoff, J. L. Marmar, G. M. Centola, I. R. Hurley. AFC 18  9 17  9 18  10 17  8
Research, Feinstein Institute for Medical Research, Manhasset, NY; Surgery, 36-40 AMH 26  22 17  12 28  1 19  14
Cooper University Hospital, Camden, NJ; Clinical Laboratory, New England AFC 13  8 12  7 14  9 11  6
Cryogenic Center, Newton, MA. 41-45 AMH 10  7 11  8 15  6 11  8
OBJECTIVE: Actin dynamics affects cell cycle progression, cell junction AFC 85 94 96 84
function, spermiogenesis and spermiation. Actin immunoreactivity is low in
testis of infertile men with varicocele. Varicocele actin loss may lead to
sloughing of germ cells, increased apoptosis, and abnormal sperm head CONCLUSION: This is the first study to estimate normative values for
shape. Testes cadmium (Cd) is elevated in infertile men with varicoceles. AMH and AFC in the general population and to compare these markers
Athough Cd can directly disassemble and degrade actin, it may also reduce across ethnicities. Latina women show lower AMH than Caucasians despite
actin by altering gene expression. In this study, we asked if Cd changed actin- similar AFC, while Chinese women have lower AFCs than African Ameri-
related gene expression in mammalian testes. cans, a finding not anticipated by the reported later age at menopause. These
DESIGN: We modeled chronic Cd exposures in Cd-sensitive rats, previ- findings suggest ethnic differences in ovarian biology that are not explained
ously reporting time and dose dependent increases in testicular Cd and by standard known confounders or available epidemiological evidence.
germ cell apoptosis, and decreased epididymal sperm counts. Now we exam- Supported by: NICHD/NIA R01HD044876 & NIH/NCRR UL1
ined expression of 105 cytoskeleton regulating genes. RR024131.

S62 Abstracts Vol. 94., No. 4, Supplement, September 2010