Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
lower LH levels in their last stimulation control than non-pregnant patients (6.5
versus 14.9 mIU/ml). A cut-off of 15 mIU/ml of LH could be calculated. The
pregnancy rate was significantly lower in patients with a LH value > 15 mIU/ml
(PR 3.2 %) compared to those with a LH ≤ 15 mIU/ml (PR 17.2 %, p < 0.01).
After ovulation induction the pregnancy rate was almost as twice as high than
after spontaneous ovulation (14.9 versus 7.4 %, n.s.).
Conclusions: It can be concluded that ovulation induction improves the probability of conception after hormonal stimulation and IUI or timed intercourse.
Awaiting occurrence of LH surge is associated with lower pregnancy rates probably due to an increased risk of post-ovulatory therapy.
P-532
Clinical results and managements for natural cycle IVF
K. Kato , T. Segawa1, S. Kawachiya1, T. Okuno1, T. Kobayashi1, Y. Takehara1,
O. Kato1
1
Kato Ladies Clinic, 7th Floor, Tokyo, Japan
Introduction: Natural cycle invitro fertilization (IVF) was most ideal way
utilized patient’s own hormonal secretion. However, control of spontaneous
ovulation is sometimes difficult in the point of view to decide optimal oocyte
collection, and usually only one oocyte is produced. We try to overcome these
difficulties many years and this time report our protocol and ingenuity of natural cycle IVF.
Materials and Methods: We investigated 2,605 cycles of natural cycle IVF
performed between January 2008 and December 2008 in our clinic. Their mean
age was 38.3 ± 5.0 years old. All cases were informed consent and trans-vaginal
ultrasound and hormonal assay (E2, LH, P4) were performed several days before expected ovulation. Dominant follicle reached 18mm in a diameter and
E2 levels reached 250pg/ml, we administrated GnRH agonist to trigger oocyte
maturation.
Three protocols for the timing of maturation triggering and oocyte retrieval
were used, depending on the LH levels. The first protocol nvolved triggering on
midnight and retrieved 32 to 34hrs later (Type A), the second involved triggering during the day and retrieved following day (Type B), and the third involved
no use of GnRH agonist and retrieved on the same day or on the following day
(Type C). In some cases, a single injection of a GnRH antagonist (0.125 mg or
0.25 mg) was used to suppress a spontaneous LH surge. All cases divided into
five groups dependent on their age (-30, 31-35, 36-40, 41-45, 46- years old) and
six groups depending on their LH levels (-9.9, 10.0-14.9, 15.0-19.9, 20.0-24.9,
25.0-29.9, 30.0-mIU/ml).
After inseminated by conventionally or intracytoplasmic sperm injection
(ICSI), normally fertilized pronuclecus zygotes were cultured individually in
20µl of cleavage medium (SAGE, USA) from day1 to day3, and in Blastocyst media (SAGE, USA) from day4 to day6. All embryo transfer performed
single transfer and diagnosed for chemical pregnancy after serum β-hCG levels above 20mIU/ml and for clinical pregnancy after confirmed gestational
sac.
Results: Spontaneous ovulation occurred in a total of 205 cases (7.9%), with no
differences according to age. Oocytes were collected in 1674 cases (69.8%) in
total, and significantly lower in the group of over 46 years old (57.1%, p < 0.01).
Fertilization and cleavage rates were 84.7% and 91.0%, and not differences
depending on age. Chemical and clinical pregnancy rates for fresh cleavage
stage embryo per transfer were 39.2% and 35.4% in total, 45.3% and 43.8%
(-30 years old), 59.1% and 48.1% (31-35 years old), 37.7% and 33.0% (36-40
years old), 20.0% and 15.0% (41-45 years old) and 0% and 0% (46- years old)
respectively. Developmental rate to blastocyst were 41.8% in total, 50.0% (-30
years old), 54.7% (31-35 years old), 53.5% (36-40 years old), 31.4% (41-45
years old) and 21.2% (46- years old) respectively.
The percentage about the timing of Type A and Type B plus C in the groups
of LH level were 94.4% and 5.6% (-9.9mIU/ml), 53.4% and 46.6% (10.0-14
.9mIU/ml), 16.1% and 83.9% (15.0-19.9mIU/ml), 6.4% and 93.6% (20.0-24
.9mIU/ml), 5.6% and 94.4% (25.0-29.9mIU/ml), 10.5% and 89.5% (30.0-mIU/
ml) respectively. In the group of LH level was 10.0 to 14.9mIU/ml, spontaneous
ovulated rate was 14.8% and significantly higher than that in the other LH level
groups (p < 0.01).
Conclusions: The rates of oocyte collection and fertilization, cleavage, and
pregnancy after natural cycle IVF were acceptable. The rate of successful oocyte retrieval was increased by optimizing the retrieval time, but further insight
is needed for the treatment of patients with slightly increased LH levels because
of the high frequency of spontaneous ovulation.
K. Jayaprakasan1, L. Nardo2, J. Hopkisson1, B. Campbell1, N. Raine-Fenning1
1
NURTURE Queen’s Medical Centre University of Nottingham, Reproductive
Medicine, Nottingham, United Kingdom
2
St. Mary’s Hospital Whitworth Park, Reproductive Medicine, Manchester,
United Kingdom
Introduction: Antral follicle count (AFC) and anti-Müllerian hormone (AMH)
are the most direct tests for quantitative ovarian reserve screening and appear
equally predictive of ovarian response during IVF/ICSI treatment. Whilst AMH
assays have recently been standardised there is no agreed technique to measure the AFC and several different ultrasound methods have been reported.
The objective of this quasi-experimental study was to compare the relationship
between AMH and antral follicle counts made using conventional 2D and 3D
ultrasound in two different centres and to analyse their ability to predict ovarian
response to controlled ovarian stimulation during assisted reproduction treatment (ART).
Material and Methods: A total of 316 subjects aged < 41 years with regular
menstrual cycles of 21 to 35 days undergoing their first cycle of ART using a
conventional long down-regulation protocol at two University-based assisted
conception unit were prospectively recruited. Transvaginal ultrasound and venepuncture were performed in the early follicular phase (day 2-5) of the spontaneous menstrual cycle immediately prior to treatment. Subjects were excluded
if they were found to have an ovarian cyst or follicle measuring ≥ 20 mm in
diameter. The number of antral follicles measuring 2-10 mm was measured using
a conventional 2D technique in one unit (Manchester) and by 3D technique in the
other (Nottingham). The main outcome measures were the relationship between
the AFC and early follicular phase AMH levels and the number of mature oocytes
retrieved. Secondary measures included prediction of poor ovarian response. The
distribution of the data was checked for normality using a normal probability plot.
The relationship of each follicular cohort with AMH and ovarian response was
evaluated using Spearman’s correlation coefficient (r). Regression analysis and
receiver operative characteristic (ROC) curve analysis was used to evaluate the
predictive value of AFC for the number of mature oocytes retrieved and poor response. Subsequently, comparison of two unpaired ROC curve areas were made.
Results: Analysis was performed on 296 subjects after excluding 20 subjects who
did not meet the inclusion criteria. The median (range) age, BMI, basal FSH and
antral follicle count were 33 (22-40) years, 24 (20-35) Kg/m2, 7.1 (3.0-14.6) IU/L
and 11 (0-42) respectively. The median (range) serum AMH level and number of
mature oocytes retrieved were 12.1 (0.9-89.3) pmol/L and 11 (6-32) respectively.
The relationship of AFC with AMH and the number of mature oocytes retrieved was similar for the measurements made with conventional 2D (0.665
and 0.519 respectively) and manual 3D ultrasound (r = 0.641 and 0.618 respectively). Multiple linear regression analysis incorporating age, FSH, AMH and
AFC revealed the better model for predicting the number of oocytes retrieved
included AFC measurements made with the 3D technique (R2 = 0.456) in comparison to the 2D (R2 = 0.416) technique. Receiver operating characteristic
curve analysis showed the predictive value of AFC for poor ovarian response
was better for the 3D technique with significantly higher area under the curve
(0.934; 0.892-0.976) compared to 2D (0.809: 0.721-0.897).
Conclusions: Antral follicle counts measured using 2D and 3D techniques
demonstrate a similar relationship to serum AMH levels and predictive value
for the number of mature oocytes retrieved in ART. However, the ability to
discriminate poor responders was better when measurements were made with
3D ultrasound technique.
POSTERS
REPRODUCTIVE GENETICS (PGD/PGS)
P-534 Incidence of aneuploidy in oocytes generated by patients with sex
chromosomal mosaicism
A. Crippa1, M.C. Magli1, F. Robles1, A. Capoti1, A.P. Ferraretti1, L. Gianaroli1
S.I.S.Me.R. s.r.l., Reproductive Medicine Unit, Bologna, Italy
1
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1
P-533 Relationship of antral follicle count with AMH and ovarian
response is similar for 2D and 3D ultrasound techniques
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-535 ESX1 gene transcript as a spermatogenesis marker in infertile men
A. Gallina1, E. Bonaparte2, M. Moretti1, G.M. Colpi3, F. Nerva3, G. Contalbi3,
L. Vacalluzzo3, S. Tabano1, F.R. Grati2, G. Gazzano4, S.M. Sirchia1,
G. Simoni2, M. Miozzo1
1
University of Milan School of Medicine, Department of Medicine Surgery and
Dentistry, Milan, Italy
2
TOMA Advanced Biomedical Assays SpA, Research and Development Cytogenetics and Molecular Biology, Busto Arsizio (VA), Italy
3
Azienda Ospedaliera San Paolo, Andrological-Urology Unit and IVF Center,
Milan, Italy
4
Azienda Ospedaliera San Paolo, Pathology Unit, Milan, Italy
Introduction: Azoospermic patients, of both the OA (obstructive azoospermia)
and the NOA (non-obstructive azoospermia) class, benefit from intracytoplasmic sperm injection (ICSI), because testicular sperm extraction (TESE) or its
variant involving multiple surgical micro-sampling of one or both patient’s
testes (microTESE) can retrieve the rare spermatozoa even in selected NOA
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cases. Identifying ongoing spermatogenesis molecular markers, predictive of
a successful sperm recovery, is therefore a valuable goal. ESX1 is a X-linked
human homeobox gene expressed in testis, placenta, brain and lung. The mouse
homologue, Esx1, is expressed in placenta and testis, specifically in pre- and
post-meiotic germ cells. In humans, ESX1 role has not been defined as yet.
Material and Methods: We have investigated ESX1 genomic sequence, expression (by RT-PCR), and epigenetic status (by promoter pyrosequencing) in
testicular tissue samples from 81 infertile subjects, to check a possible association between ESX1 alterations and impaired spermatogenesis.
Results: ESX1 gene transcript was detected in 95% of samples showing varied levels of spermatogenesis at histological analysis: in 5/6 patients with incomplete Sertoli cell-only syndrome (iSCOS), in 4/6 subjects with complete
maturation arrest (cMA), and in 100% of cases diagnosed as OA (33), hypospermatogenesis (18) and incomplete maturation arrest (iMA) (2). By contrast, it
was detected in only in 3/16 (19%) of cases histologically classified as complete
Sertoli cell-only syndrome (cSCOS). In the same patient cohort, successful
sperm recovery was obtained in 100% of OA, hypospermatogenesis and iMA
cases. In patients affected by a more severe impairment of spermatogenesis,
sperm recovery was achieved in: cSCOS (5/16), iSCOS (4/6) and cMA (2/6).
TESE was carried out in 19 of these 30 cases, and micro TESE in the remaining
11 cases. In this subset of patients, ESX1 expression and sperm extraction data
were concordant in 14/19 cases subjected to TESE (74%, p < 0.04 applying
one-tailed exact Fisher’s test), but only in 3/11 men who had undergone microTESE (27%). Concordance between ESX1 expression and sperm recovery was
therefore much higher when the twin samples subjected to ESX1 expression
analysis and sperm extraction originated from adjacent testicular areas (as it
happens in TESE). The pyrosequencing of ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors as compared to
non-expressors.
Conclusions: ESX1 was proven to be a spermatogenesis-related gene in the
human. Crossing of histological, ESX1 expression and sperm retrieval data suggested that ESX1 transcript is a sensitive indicator of spermatogenesis across
all patients, and that, in NOA patients affected by testicular heterogeneity in respect to the presence/distribution of spermatogenesis foci, discordance in ESX1
transcript detection and sperm recovery data mostly reflected the sampling procedure. Thus, ESX1 transcript emerges as a reliable spermatogenesis molecular
marker and as a predictor of gamete recovery from infertile men.
P-536 Preliminary validation of SNP genotyping and karyomapping
for preimplantation genetic diagnosis of fifty eight autosomal single gene
defects
A. Handyside1, A. Gabriel2, A.R. Thornhill1, E. Clemente3, C. Reitter3,
N. Affara3, D.K. Griffin2
1
London Bridge Fertility Gynaecology and Genetics Centre, Reproductive
Genetics, London, United Kingdom
2
University of Kent, Dept of Biosciences, Canterbury, United Kingdom
3
University of Cambridge, Dept of Pathology, Cambridge, United Kingdom
Introduction: Genome wide single nucleotide polymorphism (SNP) genotyping of parents and appropriate family member(s) allows the parental origin of
chromosomes and chromosome segments to be identified in preimplantation
embryos by Mendelian analysis, or karyomapping, providing a universal method for the combined detection of chromosome aneuploidy and linkage based
preimplantation genetic diagnosis (PGD) of single gene defects (SGDs) and
other inherited conditions. To date, about 125 inherited conditions, including
101 SGDs, have been licensed by the Human Fertilisation and Embryology Authority (HFEA) for PGD in the UK. Of the SGDs, 58 are caused by mutations in
57 different autosomal genes distributed across all 22 autosomes. Here, we examine the efficacy and accuracy of karyomapping for all 57 of these autosomal
SGDs in a couple who had PGD for a structural chromosome abnormality.
Materials and Methods: A white Caucasian couple having PGD for a structural chromosome abnormality using conventional methods consented to follow up analysis of seven biopsied unbalanced embryos by SNP genotyping and
karyomapping. After zona removal, each embryo was transferred into 2 ul of
PBS and stored at -20°C on day 4 post insemination. Following whole genome
amplification (WGA) (Genomplex, Sigma), parental genomic DNA and the
WGA products were genotyped at 299,174 genome wide SNP loci using a bead
array (Illumina CytoSNP-12, Illumina) and the SNP genotype data analysed by
karyomapping using a custom macro in Microsoft Excel. (The SNP data were
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Introduction: Sex chromosome mosaicism is not uncommon and is often seen
in individuals with ovarian dysgenesis. Data on the frequency of these aberrations in germ cells are lacking, but the incidence of sex chromosome mosaicism
has been reported to be elevated in woman with recurrent spontaneous abortions. The aim of this study was to evaluate the frequency and type of chromosomal errors in the oocytes from patients with sex chromosome mosaicism,
through the chromosomal analysis of the first polar body (PB1).
Materials and Methods: A total of 22 infertile couples underwent to 27 ICSI
cycles with PB1 analysis. Sex chromosome mosaicism was diagnosed in 8 women
(Group I, 13 cycles) with the following karyotype: 46,XX(94)/47,XXX(6);
46,XX(92)/45,X(8); 46,XX(92)/45,X(5)/47,XXX(3); 46,XX(91)/45,X(9);
46,XX(40)/47,XXX(60); 46,XX(68)/45,X(6)/47,XXX(2); and two cases
46,XX(96)/45,X(4). Fourteen couples with a normal karyotype and similar
characteristics to Group I were treated in the same period and represented the
control group (Group II). PB1s were tested for the chromosomes 13, 16, 18,
21, 22, 15 and X. The maternal age in the two groups was, 39.7 ± 4.8 years
in Group I and 38.0 ± 3.0 years in Group II (P = 0.273).
Results: The proportion of aneuploid oocytes was 45.4% in Group I and 43.8%
in Group II.
The frequency of single chromosome aneuploidy in the oocytes from Group
I and Group II were similar for chromosome 13 (3.5% vs. 7.7%, P = 0.255),
18 (9.5% vs. 16.2%, P = 0.187), 21 (10.0% vs. 15.7%, P = 0.175), and X
(16.1% vs. 14.1%, P = 0.668), whereas it was significantly higher in Group I
for chromosomes 16 (19.4% vs. 5.4%, P = 2.31E-6) and 22 (15.9% vs. 6.7%,
P = 0.0176). In three patients performing 6 cycles, the sex chromosome aberrations in the female karyotype were significantly lower compared to the frequency of aneuploidy for chromosome X detected in the analysed oocytes:
in 46,XX(94)/47,XXX(6) (2 cycles) the frequency was 25% (P = 0.007);
in 46,XX(92)/45,X(8) (2 cycles) the frequency was 30% (P = 0.002) and in
46,XX(96)/45,X(4) (2 cycles) the frequency was 25% (P = 0.003). There
were no differences in 46,XX(92)/45,X(5)/47,XXX(3) (3 cycles) where the
frequency of aneuploidy of the chromosome X was 17.6% (P = 0.160). In
the 46,XX(91)/45,X(9) (2 cycles), 46,XX(40)/47,XXX(60) (1 cycle) and in
46,XX(68)/45,X(6)/47/XXX(2) (1 cycle), the analysed oocytes (2-5 oocyte per
couple) did not present aneuploidy for chromosome X.
Conclusions: The frequency of chromosome abnormalities in infertile couples
is increased compared with the population baseline. In this study, the proportion
of aneuploidy oocytes was more of the 40% in study and control groups, but
only two chromosomes, 16 and 22, showed a significantly higher incidence of
aneuploidy in Group I compared to Group II. More important, the frequency
of chromosome X aneuploidy was exactly the same in the patients with sex
chromosome mosaicism as in the group with a normal karyotype. Furthermore,
in Group I the proportion of oocytes with aneuploidy for chromosome X was
significantly higher when compared to the frequency of sex chromosomal abnormalities in the karyotypes, where the aberrant cell lines constitute < 10% of
all analysed metaphase. These preliminary data on the oocytes confirm previous results on embryos demonstrating that when sex chromosome mosaicism in
blood lymphocytes is low (< 10% of all analysed metaphases), this condition
is not predictive of higher risk of chromosome X nondisjunction in oocytes.
Validation of these data would be especially interesting in cases where the incidence of sex chromosome mosaicism in blood lymphocytes is high.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-537 FSH-R polymorphism in severe types of OHSS (type III and
IV/V): results of a Czech pilot study
M. Macek Sr.1, P. Feldmár2, H. Klucková1, M. Hrehorcák2, J. Diblík1,
P. Paulasová1, M. Turnovec1, S. Vilímová1, M. Macek Jr.1
1
Charles University Prague and University Hospital Motol, Department of
Biology and Medical Genetics, Prague, Czech Republic
2
Charles University Prague and University Hospital Motol, Department of
Gynecology and Obstetrics, Prague, Czech Republic
Introduction: The Asn680Ser polymorphism influences the FSH-R sensitivity
to FSH and thus the risk of ovarian hyperstimulation syndrome (OHSS). Genotype Asn680/Asn680 is associated with highest, while Ser680/Ser680 with the
lowest sensitivity. Thus far, studies of FSH-R polymorphism impact on clinical severity of OHSS are infrequent with contradictory results. The aim of our
study was to compare the frequency of this polymorphism in Czech females
with severe type of OHSS versus controls.
Material and Methods: The FSH-R polymorphism genotypes were ascertained in 317 randomly selected fertile controls. These results were compared to
11 females with type III a 10 with type IV/V OHSS. Type III was characterized
by ovaries larger than 10 cm and by ultrasound-detected ascites, type IV had
ovaries larger 12 cm and clinically apparent ascites and/or hydrothorax, dyspnoe, type V had in addition to features present in type IV hypovolaemia, oliguria and hemoconcentration. Affected females with type III, IV/V had average:
BMI 25.8-23.4; age 31.5-33.0 years; estradiol 8,200–22,492 pmol/L; number of
aspirated oocytes 22-26, respectively. FSH-R polymorphism was examined by
real-time PCR on ABI Prism 7000.
Results: Controls were characterized by the prevalence of the Asn680/Asn680
(37.2%), Asn680/Ser680 (47.3%) and Ser680/Ser680 (15.5%). In type III OHSS
the prevalence of Asn680/Ser480 genotype increased from 47.3 % to 72.3%,
while Ser680/Ser680 genotype fell from 15.5 % to 0%. In type IV/V Asn680/
Asn680 increased from 37.2% to 60.0%. Due to small numbers of tested cases
compared to the higher number of controls statistics was not appropriate.
Conclusions: The results of our pilot study indicate that risk of less severe
type III of OHSS is associated with Asn680/Ser680 heterozygosity, whereas
the most severe types IV/V are associated with the highest sensitivity genotype
Asn680/Asn680. These results support the hypothesis that the FSH-R genotype
predicts not only the increased risk of OHSS, but also its clinical severity in
Czech females. Systematic study of larger cohorts is necessary to verify these
risks in other populations with different ethnicity and lifestyle
Supported by MZOFNM2005 and NR 9448-3/2007.
P-538 The FMR1 gene in human granulosa cells: expression and
functional insights
L. Fontes1, L. Haddad1, E. Borges Jr2, A. Iaconelli Jr2, D.P.A.F. Braga3,
A.M. Vianna-Morgante1
1
Institute of Biosciences University of São Paulo, Department of Genetics and
Evolutionary Biology, Sao Paulo, Brazil
2
Fertility, Assisted Fertilization Center, Sao Paulo, Brazil
3
Sapientiae Institute, Educational and Research Center in Assisted
Reproduction, Sao Paulo, Brazil
Introduction: The FMR1 (Fragile X Mental Retardation 1 gene) premutation is
the most frequent genetic factor associated with premature ovarian failure (FXPOI – fragile X-associated primary ovarian insufficiency). FX-POI is present in
about a quarter of premutation carriers. FMR1 is normally expressed in germ
cells of human female fetuses. Fmr1 mRNA is highly concentrated in mouse
ovaries at the stage of oogonia cell proliferation, during development and, in
adult female mice, Fmrp levels are higher in growing follicles. The size of the
FMR1 CGG repeat, the pattern of X-chromosome inactivation, transcription
and translation alterations, and RNA toxic gain-of-function are factors that may
be involved in the risk for FX-POI.
Material and Methods: To investigate the FMR1 gene functioning in the
human ovary, we evaluated the expression FMR1 mRNA and FMRP and subcellular FMRP distribution in human granulosa cells obtained from follicular
aspirates of women undergoing in vitro fertilization.
Results: FMR1 mRNA was detected in these cells by RT-PCR and FMRP, by
Western blotting. Various isoforms of the protein were observed, the molecular
weight of which did not differ substantially from those observed for Fmrp from
rat brain. Immunofluorescence of cells cultured for six days revealed a diffuse,
dotted, cytoplasmic distribution of FMRP. After induction of oxidative stress
by treating cell culture with sodium arsenite, FMRP was shifted to coarse perinuclear granules, colocalizing with TIA1, a stress granule marker.
Conclusions: These data indicate that, human ovaries at ovulation/hyperovulation present high expression levels of FMR1 mRNA and FMRP and, similar to
the rat and mouse central nervous system submitted to stress, human FMRP
function should be regulating translation in ovarian granulosa cells, shifting between polyribosomes and stress granules, in specific physiological conditions.
Biochemical fractionation experiments will help to validate the morphological
data reported here.
P-539 Ploidy of spermatogenic cells in testicular suspensions of nonmosaic Klinefelter’s syndrome cases, using a computerized cell scanning
system
A. Komsky1, E. Kasterstein1, D. Komarovsky1, O. Bern1, B. Maslansky1,
T. Kaplan2, A. Raziel1, S. Friedler1, Y. Gidoni1, I. Ben-Ami1, R. Ron-El1,
D. Strassburger1
1
Assaf Harofeh Medical Centre, IVF Unit, Zerifin, Israel
2
BioView, Ltd, Nes Ziona, Israel
Introduction: Klinefelter’s Syndrome (KS) is the most frequent chromosomal
abnormality in infertile men. Meiotic completion of spermatogenic cells and
the origin of euploid spermatozoa in these patients remain controversial, especially due to difficulties in the identification of these cells. Our aim was to
study the ploidy of spermatogenic cells from non-mosaic KS patients, based on
corresponding morphology and FISH images.
Material and Methods: Testicular tissue specimens were obtained from eleven
men with non-mosaic KS, six of whom had spermatozoa in their testicular tissue (positive) and the remaining five had none (negative). Three autopsy specimens from accident victims were used as a control group. Testicular tissue
was minced and enzymatically digested. Cells obtained were affixed using a
Cytospin centrifuge and alcohol. The cells were multi-sequentially stained by
Giemsa-May Grunwald, then by FISH for centromeric probes X, Y and 18. We
used a computerized automated cell scanning system (DuetTM, BioView Ltd,
Nes-Ziona, Israel) which enables stimultaneous view of morphology and FISH
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only analysed as a set of anonymous markers for Mendelian analysis purposes
and no other features such as known associations with genetic conditions or
copy number variation were analysed.) For each SGD, informative SNP loci
within 3 Mb regions flanking either side of the gene and any intragenic loci
were examined in both of the parental chromosomes inherited by six of the
embryos. The genotype of the remaining embryo was used as a reference to
establish phase.
Results: Excluding the parental translocation, 3/7 embryos had maternal aneuploidies (trisomy 16, monosomy 16, 21 and 22 and monosomy 16). Of the
57 gene loci examined, only two -FANCA (Fanconi’s Anaemia A) and ARSA
(Metachromatic Leukodystrophy) on chromosomes 16 and 22 - are outside the
region covered by the SNP loci genotyped using the chosen array. For the other
55 SGDs, an average of 217 (range 71 to 374) informative SNPs were identified
in the flanking regions of both parental chromosomes combined. Furthermore,
for 38 autosomal SGDs (69%), there were 1-27 informative intragenic SNPs.
Karyomap analysis of each locus (excluding regions of imbalance) in the six
embryos (n = 641) unambiguously identified parental haplotypes in almost all
cases (97.8%), including a significant proportion (36%) with one or more concordant intragenic informative SNPs. Of the few cases where the gene could not
be assigned to a parental haplotype, most involved a crossover between flanking
informative SNPs and in two cases an intragenic crossover. The incidence of
non-concordant informative SNPs was low (80/26,755; 0.3%). The average interval between flanking informative markers for all the loci examined was 861.2
Kb (range 52.3-5052.4 Kb). The only exception was CFTR (cystic fibrosis) on
chromosome 7, which in this couple required analysis of SNP loci further distal
to the gene to be fully informative.
Conclusions: Genome wide SNP genotyping and karyomapping unambiguously identified almost all of the parental haplotypes flanking all 55 autosomal
SGDs examined (located within the regions covered by the array). Furthermore,
the relatively short distance between flanking informative SNPs and the presence of one or more intragenic informative SNPs in many cases, minimises the
risk of misdiagnosis resulting from double recombination, eliminating the need
for mutation analysis.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-540 Assessment of male genome competence
R. Maggiulli1, D. Monahan1, Q.V. Neri1, J.C.Y. Hu1, Z. Rosenwaks1,
G.D. Palermo1
1
Weill Cornell Medical College, Ronald O. Perelman & Claudia Cohen Center
for Reproductive Medicine, New York NY, U.S.A.
Introduction: Motile Sperm Morphology examination (MSOME) is often requested by patients, and entails the live scanning of magnified spermatozoa for
irregularities linked to a compromised ability to fertilize and support embryo
development. It has also been claimed that genomic integrity is impaired in
spermatozoa displaying vacuoles and other surface irregularities. However, as
the role of chromatin fragmentation on the health of the conceptus and its ability to implant has not been confirmed, so the link between sperm head surface
defects and genomic competence has not been proven.
Here we aim to assess genomic complement and epigenetic information of
male gametes carrying surface irregularities and vacuoles.
Material and Methods: Consenting men undergoing infertility screening were
recruited. Following standard semen analysis, the sperm suspension was adjusted to 1 x 106/ml and diluted in 1:8µl PVP solution. With a micromanipulator
100 motile spermatozoa showing head dysmorphisms were selected at 6000X,
retrieved individually, and placed on pre-coated slides. These dysmorphisms
included acrosomal abnormalities as well as single or multiple vacuoles occupying at least 4% of the sperm head. Normally appearing motile spermatozoa
served as controls. Sperm DNA fragmentation was assessed by placing on a
glass slide two droplets of 3µl prewarmed agarose each containing 50 spermatozoa. Once the gel solidified, slides were immediately immersed in an acid
solution to generate single-stranded DNA, transferred to a lysing solution to
remove the nuclear proteins, dehydrated in ethanol series, and finally stained
with Giemsa (Halosperm®).
In addition, fixed spermatozoa were permeabilized with 0.1% Triton X-100
in 0.1% sodium citrate and incubated with TdT and BrdUTP for the detection
of DNA breaks. The detection of BrdU incorporation was achieved through an
Alexa Fluor 488 dye-labeled anti-BrdU antibody (APO-BrdUTM TUNEL Assay
Kit).
To determine their ploidy, fixed spermatozoa were decondensed in 10mM
DTT in 0.1M Tris-HCl pH 8 (5 min, RT), and hybridized with two sets of probe
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mixture, which contain locus specific probes for chromosomes X, Y, 13, 15, 16,
17, 18, 21, 22 (MultiVysionTM PB probe mixture, Abbott; OligoFISH™ probe
kit, One Cell System).
TUNEL and FISH slides were analyzed at 1000X under a fluorescent microscope. The scoring technician was blinded for SCD, TUNEL, and FISH.
Results: A total of 8 men (35.9 ± 4 yrs) were included in the study. The mean
sperm concentration was 79.5 ± 56.7 x 106/ml, a motility of 52.9 ± 5%, and
normal morphology of 4.0 ± 2%. The overall incidence of sperm head defect
was 57.0%. A total of 3260 spermatozoa evaluated for DNA fragmentation and
ploidy status, revealed 1936 with surface defects and 1684 without. The total
fragmentation rate detected by SCD assay was 3.9% (23/576) for vacuolated
spermatozoa and 4.5% (22/486) for the controls. When the fragmentation was
assessed by TUNEL assay, 9.8% (68/697) of spermatozoa with head defects
displayed DNA fragmentation versus 10.3% (61/592) observed in the control.
Assessment of sperm ploidy indicated a comparable proportion of chromosomal abnormalities between the two groups (1.5% vacuolated vs 1.1% control,
respectively).
Conclusions: The presence of sperm nuclear defects assessed by high magnification microscopy does not appear to correlate with the ploidy or state of the
sperm nuclear chromatin. Thus, the presence of spermiogenetic defects such
as nuclear vacuoles and other head abnormalities do not directly translate to
chromosomal imbalances or presence of DNA breakage.
P-541 Outcomes of 303 cycles on preimplantation human leukocyte
antigen typing with or without mutation analysis
C. Beyazyurek1, G.C. Ekmekci1, H.A. Tac1, N. Ajredin1, O. Verlinsky2,
F. Fiorentino3, S. Kahraman4
1
Istanbul Memorial Hospital, Reproductive Genetics Centre, Istanbul, Turkey
2
Reproductive Genetics Institute, Molecular Genetics Laboratory, Chicago,
U.S.A.
3
EmbryoGen, Molecular Genetics Laboratory, Rome, Italy
4
Istanbul Memorial Hospital, IVF and Reproductive Genetics Centre, Rome,
Italy
Introduction: Genetically inherited hemopoietic disorders and some acquired
disorders need Hematopoietic Stem Cell Transplantation (HSCT) or bone marrow transplantation for the curation of the disease. Because of the difficulty to
find a suitable donor, Preimplantation Human Leukocyte Antigen (HLA) typing with or without a single gene defect has become a promising option for the
couples having an affected child. The objective of this study is to present our
seven year experience between 2003 and 2010 on preimplantation HLA typing
with or without a single gene disorders.
Materials and Methods: Overall 303 cycles were performed for 157 couples.
249 of these were for single gene disorders (SGD) (B-Thalassemia, Fanconi
anemia, eg.) combined with HLA typing applied to 127 couples, 54 of them
were for only HLA typing for acquired diseases (ALL,AML, eg.) applied to
30 couples. Cycles performed for single gene disorders in combination with
HLA typing were B-Thalassemia (n = 224), Wiscott Aldrich Syndrome (n = 4),
Gaucher Disease (n = 4), Alpha Mannosidosis (n = 4), X–ALD (n = 3), Fanconi
Anemia (n = 3), Hurler Syndrome (n = 3), Glanzman Disease (n = 2), Hyper
IgD (n = 1) and Sickle Cell Anemia (n = 1). One blastomere was removed from
day 3 embryos by laser technique. Mutation analysis was done using either
minisequencing or restriction fragment length polymorphism (RFLP) method
combined with linked short tandem repeat (STR) marker analysis, while HLA
typing was performed using polymorphic STR markers scattered through the
HLA gene cluster.
Results: Totally 2834 blastomeres were biopsied and in 2506 (88.4%) a complete diagnosis was achieved. Embryo transfer was performed in 155 cycles
(62.2%) with SGD + HLA typing, and 39 cycles (72.2%) with HLA typingonly. Totally 68 clinical pregnancies (35%) were obtained out of 194 ET cycles.
To date, with 48 deliveries, 57 healthy and HLA compatible children were born.
18 sick children were already cured with cord blood cell and/or bone marrow transplantation. 23 children are waiting for their newborn siblings to gain
weight and grown up more to be ready for donation of stem cells.
Conclusions: The demand for the preimplantation HLA typing is increasing
tremendously since this method is an effective therapeutic tool for the curation of an affected child. PGD for HLA typing technique allows the birth of a
healthy child who is at the same time the savior of her or his sibling. Despite
the lower probability of finding suitable embryos for embryo transfer, the data
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in the same cell. Coordinates and images of all cells found on the slide were
stored digitally for subsequent analysis.
Results: A total number of 11,991 cells (mean 2,398.2 ± 1126.30 nuclei per
case) from negative KS patients, 12,387 cells (mean 2,064 ± 1380.59 nuclei
per case) from positive cases and 1,711 cells (mean 570.3 ± 187.28 nuclei per
case) from the three controls were analyzed. The rate of secondary spermatocyte and post meiotic cells (round, elongating spermatids and sperm cells)
was 1.1 ± 1.39% in the negative KS patients, 2.9 ± 3.33% in the positive ones
and 66.6 ± 6.70% in the control. Aneuploidy in the control group was equally
distributed between mitotic (4.3 ± 7.45%) and meiotic cells (4.62 ± 5.79%
for primary spermatocytes, and 4.39 ± 7.03% for secondary spermatocytes),
with a non significant decline in the post meiotic cells (1.5 ± 1.71%). In both
negative and positive KS patients the majority of the spermatogonia were aneuploid (94.9 ± 2.22 and 91.7 ± 4.54%, respectively). Similarly, primary spermatocytes showed 90.4 ± 2.14% and 85.4 ± 9.30% anueploidy. The prevalence
of aneuploidy in the secondary and post meiotic cells was significantly lower
than in the primary spermatocytes, resulting in 13.5 ± 14.51% (P < 0.002) and
24.0 ± 35.65 (P < 0.008) aneuploid cells in the positive and in the negative KS
patients respectively.
The presence of both autosomal (18) and sex chromosomes bivalents (Sex
Vesicle body) in primary diploid spermatocytes was 11.1 ± 6.84% compared to
29.3 ± 8.43% in the control group (P < 0.03). Pairing of both 18 and XY chromosomes was identified also in the aneuploid 47XXY primary spermatocytes
(17.5 ± 9.45%). There was no difference in the percentage of pairing between
the two KS subgroups.
Conclusions: The computerized cell scanning system enables to precise identification of spermatogenic cells. In both, the study and control groups, decline in
the percentage of aneuploid cells was observed after the first meiotic division.
46XY and 47XXY spermatogonia and primary spermatocytes were present in
KS patients, with and without sperm. Pairing of XY could be detected among
47XXY primary spermatocytes. Spermatogenesis in the KS patients with and
without sperm seems to be similar.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
presented in this report shows the feasibility and the practicability of PGDHLA application.
P-542 PGD for translocations: should we continue to biopsy two blastmeres?
M. Camp1, L. Hesters1, M. Le Lorc’h2, R. Frydman3, S. Romana4, N. Frydman1
Centre Hosp. Antoine Béclère, Reproductive Biology Unit, Clamart Cedex,
France
2
Centre Hosp. Necker, Genetic department, Paris, France
3
Centre Hosp. Antoine Béclère, Gynecology Obstetric Reproductive Medicine,
Clamart Cedex, France
4
Centre Hosp. Necker, Genetic Department, Paris, France
1
P-543 p27Kip1 mutant mice exhibit increase in number of maturing follicles and profound increase in multioocyte follicles (MOFs)
J. Perez Sanz1, R. Matorras2, J. Arluzea3, Y. Romin6, J. Bilbao4, N. GonzalezSantiago5, K. Manova-Todorova6, A. Koff7, J.M. Rivera-Pomar8, C. de la
Hoz-Torres3
1
University of Basque Country, Dept. Cell Biology & Histology, Bilbao, Spain
2
University of Basque Country Cruces Hospital, Dept. of Medical & Surgical
Specialities Dept of Gynecology & Obstetrics, Bilbao, Spain
3
University of Basque Country, Dept. of Cell Biology & Histology, Bilbao,
Spain
4
University of Basque Country, Dept. of Public Health & Preventive Medicine,
Bilbao, Spain
Bizkaia Technologic Park, Vakunek SL, Bilbao, Spain
Memorial Sloan-Kettering Cancer Center, Molecular Cytology Core Facility,
New York, U.S.A.
7
Memorial Sloan-Kettering Cancer Center, Cell Cycle Regulation Laboratory,
New York, U.S.A.
8
University of Basque Country, Dept. of Medical & Surgical Specialities,
Bilbao, Spain
6
Introduction: p27Kip1 is an inhibitor of cell proliferation which belongs to CKIs
group (Cyclin-dependent Kinase Inhibitors or CdK Inhibitors). p27Kip1-/- female mice are sterile but the exact mechanism by which p27 Kip1 affects ovary
function is not yet totally established. The aim of this research was: a. To look
for possible structural differences between ovaries from p27Kip1-/- and wt mice.
b. If differences are found, to find out the molecular mechanisms determining
the role of p27Kip1 in ovary development and function.
Material and Methods: Ovaries from wt and p27Kip1-/- juvenile (4 weeks) and
young adult (12 weeks) mice were fixed in paraformaldehyde, paraffin embedded and serially sectioned. Immunohistochemical staining with VASA antibody
was performed. Subsequently, the slides were scanned and digital images were
analysed using Aperio scanner and analysis software. The analysis determined:
a. Number of VASA-positive germ cells, which corresponds to the total number
of oocytes. b. Number of follicles at different stages of growth: “small” (primordial and monolayered primary), “large” (multilayered primary, secondary
and Graafian.) c. Number of multioocyte follicles (MOFs).
Results: Our results showed an increase in the number of oocytes per ovary
(twofold greater) and in the number of “large follicles”, i.e. multilayered primary and secondary (5.4 times greater) in p27Kip1-/- mice compared to wt. The
ratio “small follicles” vs. “large follicles” was also increased in p27Kip1 mutants (twofold greater) and is an important parameter to distinguish them from
wt mice. A most striking finding was the presence of an increased number of
MOFs (multioocyte follicles) in mutant mice (32.2 times greater).
Conclusions: Our results indicate a role for p27Kip1-/- in oogenesis and ovary
development. This protein is involved in proliferation, differentiation and apoptosis, major events occurring during ovarian development. Multioocyte follicles
(MOFs) are extremely rare in normal ovaries. The strikingly increased presence
of MOFs in the mutants suggests that p27 is involved in the formation and
assembly of ovarian follicles. Further studies are needed to better understand
the ovarian cellular dynamics and might be helpful to find more effective treatments for ovarian-related diseases.
P-544 Fragile sites and chromosome breakage in embryos from carriers
of structural chromosomal abnormalities determined by preimplantation
genetic diagnosis
L. Xanthopoulou1, H. Ghevaria1, A. Mantzouratou1, P. Serhal2, A. Doshi2, J.D.
Delhanty2
1
Institute for Womens Health, UCL Centre for PGD, London, United Kingdom
2
University College Hospital, Centre for Reproductive and Genetic Health,
London, United Kingdom
Introduction: Fragile sites are sites on particular chromosomes which are more
prone to chromosome breakage when exposed to conditions of DNA replication stress. Cellular mechanisms such as cell cycle checkpoints and DNA repair
monitor and determine the stability of these sites. Under conditions of stress,
instability at these regions can predispose to de novo heritable chromosome
rearrangements.
Fragile sites are expressed as a chromosome gap, where breakage can lead
to the separation of the two parts of the chromosome divided by the gap. Aberrant chromosome fragments are then generated, where the part of the chromosome distal to the gap could often be lost at subsequent cell cycle stages.
Carriers of structural chromosomal abnormalities may opt to have Preimplantation Genetic Diagnosis (PGD), whereby one or two blastomeres are
removed and tested from cleavage stage preimplantation embryos generated
through in vitro fertilization (IVF), in order to select those embryos which are
balanced or normal, aiming to establish an unaffected pregnancy.
This study looks for evidence of chromosome breakage in embryos from
such couples undergoing PGD at the UCL Centre for PGD. The aim of the study
is to assess whether the level of breakage is higher or more frequent in cases
where the breakpoints of the aberration are on or near reported fragile sites, or
on chromosomes where no common fragile sites have been reported.
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Introduction: The effects of embryo biopsy on subsequent development of embryo are still a matter of discussion. It has been showed that biopsy of single
blastomere may have a positive effect on the likelihood of blastocyst formation,
with keeping the same efficiency for the FISH diagnosis in case of PGD for
aneuploidy screening.
In our centre, two blastomeres are usually biopsied. In the aim to investigate
the feasibility of biopsying a single blastomere in case of translocations, we
have analyzed retrospectively chromosomal results obtained for each blastomere (N = 1014) in the course of 122 PGD. We have compared the diagnosis
efficiency currently obtained with two cells to the hypothetic one obtained with
a single cell.
Material and Methods: From January 2006 to December 2008, 80 couples
underwent PGD for Robertsonian or reciprocal translocations, resulting in 122
oocyte retrievals with 507 embryos available for biopsy. We have established
the number of embryos for whom: (i) one or two blastomeres were successfully
analyzed (ii) two blastomeres were discordant ie balanced and unbalanced (iii)
blastomeres failed to be analyzed. Diagnosis efficiency (E) was calculated according (Goossens et al. 2008) ie E = (D1 + D2) (1-d)/n. D1 and D2 (number
of embryos for whom diagnosis was successfully obtained from one or two
cells); d (rate of discordant embryos); n (total number of biopsied embryos).
The hypothetic efficiency if a single blastomere was biopsied has been calculated dividing by two D1 value. A Mac Nemar test was used to compare
diagnosis efficiencies.
Results: For Robertsonian translocations, among 237 biopsied embryos,
two blastomeres were successfully analyzed for 155 (65.4%) and one for 57
(24.1%). 15 embryos (6.3 %) were discordant (4 monosomies, 8 trisomies, 2
haploidies and 1 tetrasomy). 10 embryos failed to be analyzed (4.2 %). The current efficiency found was 83.8% and would significantly decreased to 72.5% if
only a single cell was available for genetic analysis (p < 0.001).
For reciprocal translocations, among 270 biopsied embryos, two blastomeres were successfully analyzed for 170 (70.4%) and one for 56 (20.7%). 9
embryos (3.3 %) were discordant (partial monosomies associated with partial
trisomies) and 16 (5.5%) failed to be analyzed. The current efficiency found
was 88.1% and would significantly decreased to 78% if only a single cell was
available for genetic analysis (p < 0.001).
Conclusion: The present investigation showed first that the biopsy of a single
blastomere would lead to an efficiency loss of 10%.
Knowing that 6.3% and 3.3% of biopsied embryos were discordant respectively in case of Robertsonian and reciprocal translocation, the risk of transferring an unbalanced embryo in these cases woul be of 3.15% and 1.65%. In view
of these results we decide to maintain the biopsy of two blastmeres in case of
translocations.
5
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-545 Meiotic segregation analysis in reciprocal translocation carriers
with embryos from preimplantation genetic diagnosis cycles
Y. Ye1, Y. Qian1, F. Jin1
1
Women’s Hospital Zhejiang University School of Medicine, Reproductive
Endocrinology, Hangzhou, China
Introduction: Preimplantation genetic diagnosis (PGD) has been offered to
carriers of reciprocal translocations (RT) to reduce the frequency of recurrent
abortions or birth of chromosomally abnormal children pregnancy loss. However, the pregnancy rate of PGD is highly related to the number of chromosomally normal embryos available for replacement and the proportion of balanced embryos varies from different type of reciprocal translocations. Thus, it
is important to assess the risk of chromosomally normal embryos for individual
patient before PGD and provide the patient informative genetic counseling. The
purpose of the present study is to explore whether the segregation patterns of
the embryos from carriers of RT are related to chromosomes involved, position
of the breakpoints, or gender of the carrier.
Materials and Methods: A total of 34 RT carrier couples underwent 41 PGD
cycles in a university-based centers for reproductive medicine. Meiotic segregation patterns of the 278 embryos were analyzed by fluorescence in situ hybridization (FISH) in their PGD cycles. For each translocation, both centric
segments (CS, arms not involved in the translocation plus interstitial segments
situated between the centromeres and the breakpoints) and the translocated segments (TS) were measured.
Results: The ratio of alternate embryos among all the embryos is 12.6% (35 of
278). There was no significant difference between the male RT subgroup and
the female RT subgroup (12.5% vs. 12.7%) regarding the proportion of the alternate embryos. And different from previous study, we found that the incidence
of alternative embryos from PGD cycles in reciprocal translocations was not
associated with acrocentric chromosomes. The alternate embryo percentage in
the pregnancy subgroup and pregnancy failure subgroup was 16.7% and 11.4%,
respectively. The proportion of alternative segregation in preimplantation embryos from PGD cycles with TS/CS ratio < 0.2 was lower than that with TS/CS
ratio > 0.6 (8.0% vs. 16.7%).
Conclusion: The incidence of alternative segregation producing normal or balanced embryos was relatively low in reciprocal translocations associated with
i326
terminal breakpoints. The results may be helpful to predict the possibility of
normal or balanced embryos for reciprocal translocation carriers before PGDFISH cycles.
P-546 Validation and preliminary clinical results of array CGH for PGS
S. Munné1, C. Gutierrez1, C. Wagner2, D. Hill3, K. Wiemer4, J. Fischer5, B.
Kaplan2, H. Danzer3, M. Surrey3, M. Opsahl4
1
Reprogenetics, n/a, Livingston New Jersey, U.S.A.
2
Fertility Centers of Illinois, n/a, Highland Park, U.S.A.
3
ART Reproductive Center, n/a, Beverly Hills, U.S.A.
4
Northwest Center for Reproductive Sciences, n/a, Kirkland, U.S.A.
5
Reprogenetics, n/a, Livingston, U.S.A.
Introduction: Chromosome abnormalities contribute to low implantation rates
and high miscarriage rates with increasing maternal age, and thus their screening previous to transfer may increase implantation rates, reduce miscarriage
rates and increase take-home baby rates. Unfortunately, differences in methodology have yielded conflicting results when day-3 biopsy and FISH analysis
have been used. The use of Comparative Genome Hybridization (CGH) combined with blastocyst biopsy and vitrification seem to provide very high implantation rates (60%) and ongoing pregnancy rates (80%). However, patients
still prefer fresh over a frozen cycle. A superior technique to FISH and CGH is
array CGH (aCGH), which provides results in 24 hours for all chromosomes per
multiple loci. The purpose of this study is to provide validation results fro this
technique as well as preliminary clinical data.
Materials and Methods: aCGH was performed on 74 cycles from 17 fertility
centers. Average maternal age was 37.3. Indications were AMA ( ≥ 38, n = 41),
recurrent pregnancy loss (RPL) (n = 22) or other < 38 years old (n = 11). A
single cell was biopsied per embryo on day 3 of development. 605 embryos
were biopsied and the cells sent to a PGD reference lab for analysis. Normal
embryos were replaced on day 5. To determine the error rate of the technique
some abnormal embryos were fully fixed and reanalyzed by FISH. To determine if pregnancy rates and miscarriage rates were improved, we compared the
results of each center with those reported for that same center by SART controlling it by age group (< 35, 35-37, 38-40, 40-42).
Results: Only 2.4% of cells did not produce amplification. Of the cells that
amplified 38.7% were euploid, and the rest were abnormal. Euploidy decreased
with maternal age from 46.1% in < 35 years, to 44.8% in 35-39 years, to 28.1%
in the 40-42 years old group (p < 0.001). After reanalyzing 84 abnormal embryos by FISH 6 were normal by FISH (7% error rate). Of the 74 cycles, we
obtained follow up data from 40 cycles past first trimester. Of those 40 cycles
20 (50%) became pregnant, compared to an expected pregnancy rate for those
centers and ages of 39.3%. In addition, of those 20 pregnancies only one miscarried (5%) compared with a 20.4% expected miscarriage rate for those centers and ages.
Conclusions: for a new technique to be validated, it should be compared to the
standard one in use. We have found that aCGH provides results in one day turnaround, with very low frequency of no results (2%), and similar error rate than
FISH (7%) even though it can analyze double number of chromosomes. This
error rate is identical than the rate of misdiagnosis expected from mosaicism
when a single cell is analyzed (Colls et al. 2007), and thus it cannot be reduced
unless biopsy is performed on day 5. The tendency observed in pregnancy and
miscarriage rates is towards an improvement of results, and thus justifies the
performance of larger randomized trials using this technique.
P-547 Inheritability of multiple offspring in families with monozygotic
twinning after art
B. Hladikova1, A. Sobek Jr.1, E. Tkadlec2, K. Kyselova1, A. Sobek1
FERTIMED, IVF, Olomouc, Czech Republic
2
Palacky University, Department of Ecology and Environmental Sciences,
Olomouc, Czech Republic
1
Introduction: ART is known to increase the number of multiple births. The
increase in dizygotic twinning (DZT) is influenced by a higher number of
transferred embryos, but the precise nature of the increased incidence of monozygotic twinning (MZT) is presently unclear. This study has focused on the
inheritance of MZT as a possible cause of this phenomenon.
Material and Methods: In a sample of pregnant women after IVF/ICSI between 2000–2008, patients with MZT (group A) were identified using US (the
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Materials and Methods: A total of 21 couples that were referred for PGD for
structural chromosomal abnormalities; 18 reciprocal translocations, one inversion, one intrachromosomal insertion, were divided into three groups: a) one
of the breakpoints was on a fragile site, b) one of the breakpoints was near (at
a position distal) a fragile site and c) no fragile sites have been reported on the
two chromosomes involved in the aberration tested for.
Each group included 7 couples in total, treated in 9, 10 and 8 cycles respectively. In general 2 blastomeres were biopsied per embryo and tested using Fluorescent in Situ Hybridization (FISH). Either two centromeric and one subtelomeric probe or one centromeric and two subtelomeric probes were used for the
diagnosis. Full follow up of the untransferred embryos was also performed.
Results: For the chromosomes where two probes were used it was possible to
determine whether there was chromosome breakage in the embryos where at
least 2 or more of the nuclei tested (from the biopsied cells and the untransferred embryo) showed evidence for breakage.
A total of 37 embryos were analysed in group A, 74 in group B and 31 in
group C. From group A, 22/37 embryos (59.5%) showed evidence for chromosome breakage, compared to 35/74 (47.3%) and 9/31 (29%) for groups B and C.
Chromosome breakage has been reported as being a fairly widespread phenomenon in human preimplantation embryos, which would explain why chromosome breakage was still seen in embryos from group C. It seems however that in
embryos from groups B and A the frequency of chromosome breaks increases,
with group A exhibiting breaks in more than half of the embryos studied. Since
at the preimplantation stages of development the cell cycle checkpoints are not
fully functional, it could be that the conditions in the culture media where the
embryos are kept cause replication stress which then cannot be fully repaired,
resulting in embryos with chromosome breaks.
Conclusion: It seems that couples carrying translocations where the breakpoints are on or near fragile sites are more likely to generate embryos with
aberrant chromosomes due to chromosome breakage and fragment loss. Consequently these couples are at a greater risk of producing unbalanced embryos, a
new element that should be considered when counseling patients.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
described as mean ± standard error of the means, odds ratios (OR) and respective confidence limits (CL).
Results: Results of the FISH analysis showed 107 cycles with chromosomal
aberrations (FISH-) and 112 with normal chromosome organization (FISH + ).
The dose of FSH and serum estradiol level did not differ between the groups.
However, the group of patients FISH + was older then the group FISH(38.05 ± 0.38 vs. 35.86 ± 0.57, respectively; p = 0.001). Oocyte yield, reflected
by the number of retrieved oocytes per number of aspirated follicles, was higher
in the FISH- group when compared to the FISH + group (62.3% vs 73,7%,
p < 0.05). Furthermore, a higher rate of oocytes with large perivitelline space
size (0.44 ± 0.05 vs. 0.33 ± 0.04, p < 0.05) and perivitelline space granularity
(0.59 ± 0.05 vs 0.44 ± 0.05, respectively, p < 0.05) were found in the FISH +
group when compared to the FISH- group. No significant differences on pregnancy, implantation and miscarriage rates were observed between the groups of
patients FISH + and FISH-. Regression analysis showed a close relationship
between the incidence of perivitelline space granularity (OR: 1.85, CL: 1.083.15, p < 0.05) and large perivitelline space size (OR: 1.58, CL: 0.92-2.73,
p < 0.05) with the odds of chromosomal abnormalities.
Conclusion: Results indicate that, similarly to previous studies, the incidence
of chromosomal abnormalities is higher in older women, indicating that PGD
is an important tool in case of age-related infertility. Furthermore, oocyte yield
as well as the evaluation of oocyte morphology in ICSI cycles, especially regarding perivitelline space, appears to be indicative of a higher incidence of
chromosome abnormalities. A hypothesis to explain such results would be that
these alterations on the perivitelline space would be related to the abnormal
polar bodies which could reflect chromosomal abnormalities on the embryos.
However, further studies are necessary to test this hypothesis.
P-549 Diminished ovarian reserve affects embryo multiple abnormalities
occurrence in ICSI cycles
P-548 Impact of oocyte morphology on chromosomal aberrations and
ICSI outcomes
M. Nichi1, R.C.S. Figueira2, D.P.A.F. Braga3, A.S. Setti3, A. Iaconelli Jr.4, E.
Borges Jr.4
1
University of Sao Paulo (USP), Animal Reproduction, Sao Paulo / SP, Brazil
2
Fertility - Assisted Fertilization Center, IVF Laboratory, Sao Paulo / SP, Brazil
3
Fertility - Assisted Fertilization Center, Scientific Research, Sao Paulo / SP,
Brazil
4
Fertility - Assisted Fertilization Center, Clinical Department, Sao Paulo / SP,
Brazil
Introduction: The introduction of the intracytoplasmic sperm injection (ICSI),
which requires the removal of cumulus cells, allowed the visualization of new
information regarding oocyte morphology. However, it is still a matter of debate
whether morphology is a good predictor of oocyte viability. With the advent of
preimplantation genetic diagnosis (PGD) associated to techniques such as the
polimerase chain reaction (PCR) and the FISH (Fluorescent “In situ” Hibridization), for gene and chromosomal abnormalities, respectively, the selection of
the most competent oocyte or embryo became much more accurate. The FISH
is a technique that uses fluorescent probes that bind to specific parts of the chromosome in order to detect and localize the presence or absence of specific DNA
sequences on specific chromosomes. However, the practical and inexpensive
application of morphological evaluation should not be discharged with no further investigation. Therefore, this study was designed to verify the relationship
between oocyte morphology and chromosomal abnormalities evaluated by the
FISH in patients submitted to ICSI.
Material and Methods: The population in the present study included 654
embryos from 219 patients submitted to PGD following ICSI. The blastomeres were evaluated using the FISH, which was performed by spreading the
nuclei from the cells onto microscope slides, dehydrating and staining with
specific fluorescent probes. Chromosomes 13, 18, 21, X and Y were tested
for aberrations. Patients’ age, FSH administered for ovarian stimulation,
serum estradiol levels, oocyte yield, oocyte morphology (i.e., cytoplasmic
granularity, smooth endoplasmic reticulum clusters, vacuoles, perivitelline
space size, perivitelline space granularity and fragmented first polar body)
and ICSI outcome were compared among groups showing normal (FISH-)
or abnormal chromosomes (FISH + ) using the Student t test. Regression
analysis was performed to assess the relationship between studied response
variables and the incidence of chromosomal abnormalities. Results were
S.S. Colturato1, A.S. Setti2, R.C.S. Figueira1, D.P.A.F. Braga2, A. Iaconelli Jr.3,
E. Borges Jr.3
1
Fertility - Assisted Fertilization Center, IVF Laboratory, Sao Paulo / SP, Brazil
2
Fertility - Assisted Fertilization Center, Scientific Research, Sao Paulo / SP,
Brazil
3
Fertility - Assisted Fertilization Center, Clinical Department, Sao Paulo / SP,
Brazil
Introduction: A reasonable percentage of women undergoing infertility treatment respond poorly to the ovarian stimulation protocol. Low follicular recruitment following ovarian stimulation may be observed in 9 to 24% of the in vitro
fertilization (IVF) cycles. It is well known that ovarian poor response to stimulation protocols increases as women get older. Those patients are usually stimulated with high doses of gonadotropins; however, due to the low number of
retrieved oocytes and transferred embryos, low pregnancy rates are expected. In
addition, embryos originating from poor prognosis IVF patients were proposed
to have higher aneuploidy rates. The aim in this study was to test the hypothesis
that aged women with poor ovarian response express an increase on embryo
chromosomal alterations when compared to aged women who presented normal
response.
Material and Methods: The present study included couples undergoing intracytoplasmic sperm injection (ICSI) cycles associated to preimplantation
genetic screening (PGS), as a result of advanced maternal age. Patients were
subdivided into two groups according to the number of collected oocytes after
ovarian stimulation: Poor Responder (PR group, n = 34), patients who produced
four or less oocytes; and Normoresponder (NR group, n = 50), patients in which
five or more oocytes were retrieved. After ICSI, all the biopsied embryos were
analysed for chromosomes X, Y, 13, 16, 18, 21 and 22. Groups were compared
regarding ICSI outcomes and aneuploidy frequency. The presence of two or
more chromosomal abnormalities was characterized as multiple abnormalities.
Influence of poor ovarian response on aneuploidy rates was assessed using logistic regression analysis, and the results were expressed as odds ratios (OR),
confidence intervals (CI) and p-values.
Results: Cycle’s general characteristics were similar between the groups. There
were no significantly differences on fertilization (73.9% vs 76.6%; P = 0.7861),
implantation (22.6% vs 20.6%; P = 0.6863) and pregnancy rates (28.0% vs
29.4%, P = 0.9208) between NR and PR groups; however, a significantly increase on the mean number of transferred embryos (2.0 ± 1.0 vs 1.1 ± 0.8,
P = 0.0047)) was observed on NR group. Regression analysis showed no
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number of gestational sacs was higher than the number of transferred embryos
or monochorioidinity or monoamniality was clearly defined). Group A was
compared with families that only had one child delivered (group B). All patients
received a genetic questionnaire or they were personally interviewed on the history of MZ or DZ twins in their immediate family (parents, grandparents, all
siblings and children). Statistical analysis was done by chi-square test.
Results: In group A, 17 of 20 couples with MZT responded to our questionnaire (85%).
A record of MZT in the immediate family was found in 7 couples (41.2%
p < 0.001) and DZT in 4 families (23.5 %, NS). MZT or DZT (in 4 of them
the zygosity was not specified because of abortion, early child death or missing
information) were reported in 13 families (76.5 %, NS). In group B (n = 72), we
found 9.7% MZT, 22% DZT and 31% incidence of all multiple pregnancies.
Seven families who reported MZT were tested in detail. In all seven pedigrees, 51 pairs were monitored. For these couples, we found 19 families having
MZT (37 %).
Three genealogical trees, describing female and male transfer will be presented. One of our male patients had MZT with a first and also later with a
second woman.
Discussion: The frequency of MZT in families with a history of MZT is significantly higher than in families with just single child births. Autosomal dominant
inheritance with low penetrance (38%), described by Hamamy et al. (2004),
was also acknowledged in our study. Our results were in accordance with inheritance of the gene responsible for MZT, described through both maternal and
paternal lines (Harvey et al., 1977).
Conclusion: Our data suggest that monozygotic twinning has autosomal dominant inheritance across both paternal and maternal lines with low penetrance.
It is inheritance and not ART technologies that is responsible for the high
frequency of MZT after IVF.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-550 Potential benefits of PGS in recurrent implantation failure: a
prospective controlled trial in a properly characterized population
C. Rubio1, J. Domingo2, L. Rodrigo1, A. Mercader1, M.J. De los Santos3,
T. Pehlivan4, E. Bosch5, M. Fernández6, C. Simón5, J. Remohí5, A. Pellicer5
1
IVI Valencia, PGD, Valencia, Spain
2
IVI Las Palmas, Medical Reproduction Unit, Las Palmas, Spain
3
IVI Valencia, IVF, Valencia, Spain
4
IVI Istanbul, IVF, Istanbul, Turkey
5
IVI Valencia, Medical Reproduction Unit, Valencia, Spain
6
IVI Sevilla, Medical Reproduction Unit, Sevilla, Spain
Introduction: In recent years great controversy has emerged regarding the application of preimplantation genetic screening (PGS) for different indications.
Methodological aspects and patients´ selection criteria could explain differences among authors. The aim of this study was to evaluate the usefulness of
PGS in patients < 40 years with recurrent implantation failure. Patients were
prospectively randomized to either of the following two groups: conventional
IVF/ICSI or PGS, both treatments with day-5 embryo transfer.
Material and Methods: Study design: Before starting the cycle, patients were
allocated through computer-generated randomization into two groups: conventional IVF/ICSI cycle (group A) or PGS cycle with screening for chromosomes
13, 15, 16, 17, 18, 21, 22, X and Y (group B). All patients recruited underwent
an exhaustive infertility work-up: vaginal ultrasound (hysterosonography or
hysteroscopy when needed); blood karyotypes; Anticardiolipin and lupus anticoagulant antibodies; Antitrombin III and APCR levels, factor V Leiden mutation; levels of protein C and S; Serum homocysteine and screening for MTHFR
and factor II mutations. Inclusion criteria were: women < 40 years, with ≥ 3
implantation failures in previous cycles with the transfer of at least two good
quality fresh embryos per cycle. Exclusion criteria were: any abnormality in
the previous infertility work-up; hydrosalpinx; any polip or myoma; previous
ectopic or uterine miscarriages; previous difficult embryo transfer with high
difficulty and/or bleeding; other indications for PGS/PGD.
Interventions: Similar protocols for ovarian stimulation and IVF were applied in both groups. Embryos were co-cultured on a monolayer of epithelial
endometrial cells and were transferred on day-5 of development. In group B,
single blastomere embryo biopsy was performed on day-3 using a non-contact
laser system (OCTAX, Herbron, Germany) and aneuploidy was assessed by
fluorescence “in-situ” hybridization using additional rounds with subtelomeric
probes (Vysis Inc., Downers Grove, Il, USA).
Sample size calculation and interim analysis: Primary end-points were ongoing pregnancy and delivery rates and secondary end-points were blastocyst
rates, chromosomal abnormalities and ongoing implantation rates. Sample size
was calculated for a 20% difference (20 to 40%) in ongoing pregnancy rates per
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cycle between the two groups and at least 64 patients/arm with ovum pick-up
had to be enrolled in this trial (Error α = 5%; error β = 20%). The study was initiated in 2004 and an interim analysis was performed in December 2009, with
half of the patients recruited. A total of 132 patients were initially informed
about the study: 25 rejected it, 26 did not meet the inclusion criteria, 11 were
excluded after the infertility work-up and finally 70 were enrolled, 35 in each
arm. Fisher´s exact test was done to compare percentages and Mann-Whitney
test for non-categorical variables.
Results: The interim analysis revealed that patients in groups A and B had similar mean age (35.0 ± 2.8 and 35.0 ± 4.0); mean number of MII oocytes (9.9 ± 5.1
and 10.0 ± 4.8); mean number of blastomeres on day-3 (7.1 ± 1.8 and 7.7 ± 1.3)
and blastocyst rates (60.1% vs. 69.1%). No statistical differences were found
neither in the mean number of embryos transferred (1.7 ± 0.8 vs. 1.4 ± 0.8),
nor in the percentage of cycles reaching embryo transfer (88.6% vs. 85.7%) in
groups A and B, respectively. The percentage of chromosomal abnormalities
in group B was 60.9% (112/184). The one-sided analysis revealed significant
increases in group B compared to group A, regarding ongoing pregnancy rate
per transfer (50.0 vs. 25.8; p = 0.0456) and ongoing implantation rate (36.0 vs.
16.9; p = 0.0202). Group B also showed superiority in ongoing pregnancy rate
per cycle, without reaching statistical significance (42.9 vs. 22.6; p = 0.0630).
Conclusions: PGS could be beneficial in a selected group of patients with recurrent implantation failure in whom other potential causes were discarded.
Higher embryo aneuploidy rates than the expected according to patients’ age
were observed.
P-551 Spontaneous abortion and 44-bp insertiondeletion polymorphism
of the SLC6A4 gene
B. Perez-Nevot1, A.M. Lendinez1, A.R. Palomares2, M. Polo3, A. Rodriguez4,
A. Reche5, M. Ruiz-Galdón6, A. Reyes-Engel6
1
Hospital Clínico Universitario Virgen de la Victoria, Análisis Clínicos,
Malaga, Spain
2
Instituto de Fertilidad Clínica Rincón, Bioquimica y Biología Molecular,
Malaga, Spain
3
Hospital Clínico Universitario Virgen de la Victoria, Anatomía Patológica,
Malaga, Spain
4
Hospital Clínico Universitario Virgen de la Victoria, Ginecologia y Obstetricia, Malaga, Spain
5
Hospital Materno Infantil. Carlos Haya, Ginecologia y Obstetricia, Malaga,
Spain
6
Facultad de Medicina. Universidad de Málaga, Bioquimica y Biología Molecular, Malaga, Spain
Introduction: Although the relationship between antidepressant use during
pregnancy and its adverse effects has been widely investigated, very few studies
have evaluated the impact of antidepressant use during pregnancy on the risk of
spontaneous abortion (SA). Several studies suggests that gestational exposure
to antidepressants, especially selective serotonine reuptake inhibitors (SSRI),
can lead to spontaneous abortion.
On the basis of these reports we have analyzed the allele and genotypes frequencies of the 44-bp insertiondeletion polymorphism in 5-Hydroxy-tryptamine
transporter gene promoter region (5-HTTLPR ) in samples from SA.
Material and Methods: Parafine embebed tissues samples, from SA of unknown etiology (n = 29), and a group of 89 fertiles women, were genotyped
for the 44-bp insertiondeletion polymorphism located in the promoter region
of the SLC6A4 gene. Parafine from tissues was discarded by Xylol washing.
DNA was extracted from tissues and blood samples by salting-out method. PCR
amplified samples were analysed by Snapshot (Applied Biosystem).
Results: The genotypes and allele frequencies of the groups were: SA,SS = 0,71,
SL = 0,18 and LL = 0,11; control group, SS = 0,32, SL = 0,43, LL = 0,25
(p < 0,001 ). It has been reported that cells homozygous for the L form produced
steady-state concentrations of SLC6A4 mRNA that were 1.4 to 1.7 times those
in cells containing 1 or 2 copies of the S variante. In these studies, the data associated with the S/S and L/S genotypes were similar, whereas both differed from
the L/L genotype, suggesting that the polymorphism has more of a dominantrecessive than a codominant-additive effect. Although S/L polymorphism of the
SLC6A4 has been related to several mental diseases, and human behavior, and
the short (S) form (functionally L/S or S/S) shows an increase in brain metabolism, very few of these studies are referred to the develoment of the brain embryo, and none has been related to foetal viability as the present report.
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significant influence of poor response on the percentage of embryos showing
autosomal aneuploidy (OD: 1.23, CI: 0.74-2.05, P = 0.417), sexual aneuploidy
(OD: 0.9, CI: 0.44-1.85, P = 0.769). On the other hand, poor response was associated with a significantly increased risk of embryo multiple abnormalities
occurrence (OD: 2.15, CI: 1.15-4.04, P = 0.017). In addition, cycle cancelation
rate was significantly higher on PR group (4.0% vs 23.5%, P = 0.0128). This
finding was confirmed through the logistic regression model (OR: 7.38, CI:
2.35-23.24, P = 0.016).
Conclusion: In this study we attempted to associate ovarian poor response with
increased occurrence of embryo chromosomal alterations in aged women. Results showed that embryo multiple abnormalities occurrence risk is more than
two-fold higher in PR patients. This finding also makes plain the statistically
lower number of transferred embryos on PR group, since a diminished number of viable embryos were available to be transferred. In addition, odds of
cycle cancellation rate were more than seven-fold higher due to the decreased
presence of normal embryos on PR group. Similar implantation and pregnancy
rates in NR and PR groups could possibly be explained by equivalent embryo
morphology and chromosomal status of transferred embryos after PGD performance. Despite the poor prognosis of the IVF outcome in aged patients with
low response to ovarian stimulation, the present study shows that poor response
is not constantly associated with a decreased implantation and pregnancy rate,
however, poor ovarian response impairs embryo chromosomal integrity and
percentage of viable embryos, reducing the chances of IVF cycles completion.
Moreover, our results highlight the predictive value of aged women ovarian
reserve for oocyte quality and competence.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
Conclusions: Although this is an ongoing study, with a low sample number
by now, we observed a strong presence of SS genotype in samples of fetuses.
These results permit to conclude that metabolism of serotonine has some influence on human fetal viability. Supported by Grants AF2008-03314 and PTQ
09-01-00496.
P-552 Validation of putative CNVs on the X chromosome in POF patients
E.A.H. Knauff1, H.M. Blauw2, K. Kok3, C. Wijmenga3, B.C.J.M. Fauser1,
L. Franke3
1
University Medical Center Utrecht, Reproduction & Gynaecology, Utrecht,
The Netherlands
2
University Medical Center Utrecht, Neurology, Utrecht, The Netherlands
3
University Medical Center Groningen, Genetics, Groningen, The Netherlands
P-553 Parthenogenetic activation of human oocytes as a model for polar
bodies-PGD
A. Paffoni1, V. Paracchini2, S. Ferrari1, L. Restelli1, D.A. Coviello2,
C. Scarduelli1, M. Seia2, G. Ragni1
1
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milan Italy,
Department of Obstetrics and Gynaecology Infertility Unit, Milan, Italy
2
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milan Italy,
Medical Genetics Laboratory., Milan, Italy
Introduction: Pre-implantation genetic diagnosis (PGD) has become an established clinical approach for prevention of genetic disorders and polar bodies
analysis instead of, or together with, blastomeres analysis has gained increasing
importance in recent years.
We evaluated a model of parthenogenetic activation of human oocytes in
order to assess the feasibility of PGD for monogenic disorders on human oocytes in our laboratory. Aim of this model was to test an ethical experimental
setting independent from fertilization, resembling the dynamics of chromosome segregation and crossover during meiosis. In this model, after the molecular analysis of the first polar body (PB1), the second polar body (PB2) can
be obtained in order to have a complete genetic analysis and validate molecular
P-554 Application of mild stimulation and single blastocyst transfer
with vitrification in preimplantation genetic diagnosis (PGD) cycles: case
report
N. Aoyama1, Y. Takehara2, S. Kawachiya2, T. Kuroda1, N. Kawasaki1, R.
Yamadera1, T. Suzuki1, K. Kato2, O. Kato2
1
Kato Ladies Clinic, Perinatal Genetics Division, Tokyo, Japan
2
Kato Ladies Clinic, Tokyo, Japan
Introduction: The efficacy of mild stimulation and single blastocyst transfer
has been reported previously, however these techniques are not commonly applied in PGD cycles. We set out to develop a new approach for PGD.
Material and Methods: Nine couples consisting of subjects with a mean age
of 35.1 years who were carriers of translocations, had experienced two or more
consecutive pregnancy losses, and failed to experience a live birth in their entire
reproductive history. Prior to the commencement of the study, approval from the
ethical aspect was received from the Japan Society of Obstetrics and Gynecology and our local Institutional ethics committee.
From January 2007 to December 2009, 9 patients underwent mild ovarian stimulation, which was carried out by administration of clomiphene citrate
in combination with a minimum amount of urinary HMG (150IU X less than
4 times) or recombinant FSH (150IU X less than 3 times). Administration of
clomiphene citrate was initiated from day 3 of the menstrual cycle at 50mg/day
and was continued until the day before administration of the GnRH agonist as
the maturation trigger (GnRHa nasal spray, 300µg) and 32-35 h before oocyte
retrieval as described by Kato.
IVF was performed for all oocytes, and embryos reaching at least the 6-cell
stage on day 3 of development were biopsied by mechanical and cleavage aspiration.
Two blastomeres were removed and fixed by the Carnoy method. Culture of
all the biopsied embryos was continued, and embryos developing to the blastocyst stage were vitrified. FISH analysis was performed on all blastomeres using
3 to 4 appropriate FISH probes for several loci divided by the patient’s own
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Introduction: Premature ovarian failure (POF) is the spontaneous cessation
of the menstruation before age 40 accompanied with elevated FSH, low AMH
and oestradiol levels. Copy number variants (CNVs) are submicroscopic deletions or duplications ranging from 1 kilobase to several megabases and are of
increasing interest in explaining human genetic variation. Since microscopic X
chromosomal abberations identified by karyotyping are associated with POF
we hypothesized that submicroscopic CNVs also contribute to POF pathogenesis. These variants will go undetected by conventional karyotyping but can
be readily detected with high-density SNP arrays. Using Illumina 370k SNP
genotyping chips and Using PennCNV calling software, we identified putative deletions in the Xq21.3 locus in nearly 15% of the 110 patients that were
analyzed and none in controls (ESHRE abstract 2009 - Hum. Reprod. 24:i12
O-029). Notably, karyotypic abnormalities have been reported in this locus to
be associated with POF.
Material and Methods: To substantiate this finding we re-analyzed 13 of 15
subjects that suggested a deletion on the Xq21.3 locus using an 8-plex 60K
array that was custom designed using eArray. The array (ID 023317, Agilent
Technologies Inc., Santa Clara, CA, USA) contains 48,325 probes, evenly distributed over the X chromosome, with a medium distance between the probes of
1.9 Kb. All probes were selected from the probe groups present on the catalogue
1M and 400K arrays (ID 021529 and ID 021850, respectively, Agilent).
Results: We did not observe deviating intensity values for consecutive probes
in the analyzed samples, indicating that the samples do not contain CNVs in this
locus. Therefore, the putative CNVs identified are most likely false-positives.
We hypothesize that batch or probe binding effects may have played a role in
the earlier Illumina experiment.
Conclusions:
- The earlier identified deletion locus on Xq21.3 could not be validated
using a custom designed ultra high density X chromosome comparative
genomic hybridization array.
- CNVs on the X chromosome do not play a major role in the aetiology
of POF.
- Careful validation is required when using high density genotyping chips
for CNV detection.
results. Indeed, PB2 analysis is useful to confirm a diagnosis of homozygous
PB1 or to predict which maternal allele will be present in the oocyte in case of
heterozygous PB1.
Material and Methods: After partial zona pellucida dissection, PB1s were removed from spare donated metaphase-II oocytes and immediately transferred to
lysis buffer for subsequent molecular analysis. Parthenogenetic activation was
conducted on biopsied oocytes through exposure to 5 mM ionomycin in culture
medium for 5 minutes at 37 °C, 5% CO2 in the dark. Oocytes were then washed
three times in fresh cleavage medium, placed separately in 40-µL microdrops of
the same medium under mineral oil, and cultured in standard conditions. After
18–20 hours, oocytes were evaluated for signs of activation. Oocytes showing
one enlarged pronucleus and extrusion of PB2 were considered activated; PB2s
were biopsied and treated as PB1s.
The molecular analysis involved a fluorescent multiplex polymerase chain
reaction (PCR) of highly polymorphic short-tandem repeat (STR) markers,
closely linked to the disease-causing gene. In this set of experiments, a panel of
ten highly polymorphic STR markers flanking ‘Cystic Fibrosis Transmembrane
conductance Regulator’ (CFTR) gene was selected for haplotype analysis.
Based on three or more informative markers, genetic analysis was performed
on PB1 and PB2.
Results: Twenty-one oocytes were selected from seven women. After PB1 removal, twenty of them were subjected to parthenogenetic activation, and fifteen
(75%) extruded the PB2. All of PB1s were successfully PCR-amplified but three
out of 15 PB2 did not get any amplification product. Limiting the results to 12
oocytes with both PB1 and PB2, 10 out of 12 PB1s were heterozygous and 2
homozygous for the evaluated markers, thus resulting in a percentage of crossover recombination of 83%. PB2s analysis consistently permitted the prediction
of the oocyte’s genotype in case of heterozygous PB1s (n = 10), and confirmed
PB1 results in homozygous cases (n = 2).
Among all evaluated markers, we observed a frequency of allele drop out
(ADO) of 5.1% (3/59) with no more than one case of ADO for single PB.
Conclusions: The model of parthenogenetic activation confirms that polar
bodies-PGD for single gene disorders is feasible and offers a valuable tool for
setting up PGD procedures without wasting zygotes. Our results confirm a high
recombination rate in CFTR gene suggesting a very limited possibility of selecting oocytes according to genetic analysis of PB1 only.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-555 Spindle and chromosome configuration of human in vitro matured oocytes after vitrification: effect of cytochalasin b pretreated
Q.H. Xu1, Z.G. Zhang1, P. Zhou1, Z.L. Wei1, D.K. Huang2, Q. Xing3, Y.X. Cao3
1
The First Affiliated Hospital of An Hui, IVF Center, Hefei, China
2
Anhui Medical University, Basical Medical Department, Hefei, China
3
The First Affiliated Hospital of An Hui, Basical Medical Department, Hefei,
China
Introduction: Although many advances in oocyte cryopreservation have been
made, unlike embryo and sperm cryopreservation, it is still under debate, mostly because the fertilization rate and implantation rates are still low; the subsequent developmental competence is poor. Now many reports have proved that
the disappoint success rates of oocyte cryopreservation due to the damage on
the ultrastructure of human oocytes during cryopreservation. The objective of
the present study was to investigate the effect of Cytochalasin B on the spindle
and chromosome configuration of human in vitro matured oocytes after vitrification.
Material and Methods: Immature oocytes which were collected in intracytoplasmic sperm injection (ICSI) cycles (include stage GV and MI) after ovarian
stimulation, followed in vitro matured(IVM) for 24h-48h, 170 oocytes were
matured(with the extrusion of first polarbody), were randomized into five
groups to vitrification. Group A: 37 oocytes were treated with CCB for 20mins
before vitrification; Group B: 38 oocytes with CCB for 30 mins. Group C: 33
oocytes with CCB for 40 mins. Group D: 36 oocytes without CCB before vitrification. Group E: 26 ooctyes whitout vitrification and CCB as the controlled
group. 71 in vivo matured oocytes were collected from ovarian stimulation of
in vitro fertilization-embryo transfer (IVF-ET) cycles and ICSI cycles, and randomized into four groups for vitrification. Group A1: 17 oocytes treated with
CCB for 20mins before vitrification; Group B1: 20 oocytes with CCB for 30
mins. Group C1: 17 oocytes with CCB for 40 mins. Group D1: 17 oocytes without CCB before vitrification. All the oocytes were thawed after three weeks and
then immunostained for tubulin and chromatin and at last visualized by confocal laser scanning microscopy.
Results: No significant difference of survival rate among group A, B, C, D
(80.39%, 64.86%, 55.36% and 78.79%, P > 0.05). No significant difference
in survival rate among group A1, B1, C1, D1 (64.7%, 50.0%, 64.7% and 70.6%,
P > 0.05). There was no significant difference in the frequencies of normal spindle and chromosome in group A, B, C, D (21.05%, 26.3%, 27.78% and 23.81%,
P > 0.05), but significantly lower than group E (61.54%, P < 0.05). There was
i330
no significant difference in the frequencies of normal spindle and chromosome
in group A1, B1, C1, D1 (27.3%,30.0%,45.5% and 33.3%, P > 0.05). No statistical difference was found in survival rate and frequencies of normal spindle
and chromosome between in vitro matured groups treated with CCB and in vivo
matured groups after vitrification(P > 0.05).
Conclusions: Spindle and chromosome configuration of human in vitro matured oocytes was damaged in vitrification, which can not improved by CCB
pre-treatment before vitrification. Oocytes in vitro matured or in vivo matured
have the same tolerance to the cryoinjury.
P-556 Modulation of an imprinted gene network in placenta as a compensatory mechanism for normal development of in vitro manipulated
mouse embryos
P. Fauque1, M.A. Ripoche2, J. Tost3, L. Journot4, P. Jouannet1, D. Vaiman2, L.
Dandolo2, H. Jammes5
1
Hospital Cochin University Paris Descartes, Biology of Reproduction, Paris,
France
2
Institut Cochin INSERM U1016 CNRS (UMR 8104) University Paris Descartes, Department of Genetics and Development, Paris, France
3
CEA-Institut de Génomique Centre National de Génotypage, Laboratory for
Epigenetics, Evry, France
4
Institut of Functional Genomic, IFG, Montpellier, France
5
INRA - ENVA UMR 1198, Biology of Development and Reproduction, Jouy en
Josas, France
Introduction: Genomic imprinting regulates the expression of several genes
crucial for mammalian development, by parental-specific mechanisms such as
allele-specific DNA methylation at differentially methylated regions (DMRs).
We have previously shown that in vitro fertilization and embryo culture result
in methylation defects at the imprinted H19-Igf2 locus in the mouse blastocyst
(Fauque et al. 2007). The current study was designed to evaluate the consequences of the same manipulations on genomic imprinting after implantation
using a mouse model.
Materials and Methods: Mouse blastocysts were produced following three
different experimental conditions: (i) a control group with embryos obtained
after in vivo fertilization and development and two groups of embryos maintained in different culture systems (either M16 or G1/G2 medium) after (ii) in
vivo fertilization or (iii) in vitro fertilization. All blastocysts were transplanted
into pseudopregnant females. Embryos and placentas were then collected from
recipient females at day 10.5 of development. DNA methylation patterns of
the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. Furthermore the expression levels of 19 genes belonging to the
imprinted gene network (IGN) involved in embryonic growth (Gabory et al.
2009; Varrault et al. 2006) were analyzed in placental tissues using quantitative
RT-PCR and geNorm quantification.
Results: No significant alteration was observed in the methylation profiles of
four DMRs from the H19, Igf2, Igf2r and Dlk1 genes, neither in placentas nor
in embryos of experimental groups compared to controls.
Interestingly, placentas displayed a correlated misregulation of several
imprinted genes. Significantly disturbed levels of H19 and Igf2 mRNA were
observed, as well as of most other genes from the IGN. Both maternally and
paternally expressed imprinted genes were modified and mostly up-regulated.
In addition, we observed that the expression patterns were not significantly different between both culture systems.
Conclusions: In contrast to the methylation defects detected at the blastocyst
stage, we found that after in vitro manipulations, retransplanted E10.5 embryos
and placentas were phenotypically normal and showed no modification of the
methylation profiles of several imprinted genes. We hypothesize that a completely restored epigenetic pattern could be required for normal foeto-placental
development. Furthermore, the modulation of a coordinated network of imprinted genes could result in a compensatory process capable of correcting potential dysfunction of placenta and protecting the embryo. These data obtained
at post-implantation stage show a moderate risk of epigenetic abnormalities in
the mouse and appear more reassuring for the health of children born by ART
compared to studies investigating preimplantation embryos.
References:
1
2
3
Fauque P et al., 2007. BMC Dev Biol 7:116.
Gabory A et al., 2009. Development 136(20):3413-3421.
Varrault A et al., 2006. Dev Cell 11(5):711-722.
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breakpoint. Cryopreserved blastocysts which showed alternate were thawed and
intrauterine transfer of single blastocysts was performed.
Results: Sixty-eight oocytes were collected in 25 OR cycles from 9 patients,
and 94% (60/64) were fertilized, 95% (57/60) of the embryos were biopsied,
and 100% (57/57) of the embryos were successfully biopsied. Of the embryos
that were successfully biopsied, 98% (56/57) yielded a diagnostic result, of
which only 30% (17/56) were transferable at the 8-cell stage. Culture of all the
biopsied embryos was continued and 61% (35/57) of the embryos developed to
the blastocyst stage. Of the 35 vitrified blastocysts, only 37% (13/35) resulted
in alternate, and 11 of the blastocysts which showed alternate were thawed and
intrauterine transfer was performed by single blastocyst transfer for 8 patients.
Two blastocysts are still cryopreserved and kept for second children. A positive
heart beat in 7 cycles was obtained in 7 patients, 64% per ET (7/11) and 28%
per OR (7/25).
Finally, the delivery rate was 55% per ET (6/11) and 24% per OR (6/25),
and one case is ongoing at 9 weeks; there were no spontaneous abortions and
none of the clinical pregnancies was lost to follow up. There were 6 deliveries
of 6 babies, consisting of 2 males and 4 females with a birth weight of 2198
to 3296g and gestational age of 37.0 to 40.4 weeks, with no malformations at
birth. Prenatal diagnosis revealed normal karyotype in 3 and inherited reciprocal translocation in 2 cases. Pre or post-natal diagnosis was not undertaken in
the remaining one case, however, there were no reports of developmental or
mental retardation in any of the children. Therefore, we assume that all of the
babies were healthy.
Conclusions: Many researchers have reported that the most important factor
for PGD is the risk of misdiagnosis. The PGD results of 9 patients in this study
strongly suggest that not only the diagnosis was performed accurately, but also
the mild stimulation and single blastocyst transfer with vitrification was effective for PGD.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-557 Application of A-CGH on blastocyst biopsy in patients with
idiopathic recurrent abortions
A. Hellani1, A. Elsheikh1, K.K. Abuamero2, S. Elakoum3
1
Saad Specialist Hospital, PGD Laboratory, Alkhobar, Saudi Arabia
2
King Saud University, Genetics, Riyadh, Saudi Arabia
3
Saad Specialist Hospital, PGD Laboratory, Elkhobar, Saudi Arabia
P-558 Association of folate-pathway gene polymorphisms with pregnancy outcome in recipient women undergoing in vitro fertilization with
donor eggs
A.R. Palomares1, A.M. Lendinez2, B. Perez-Nevot2, F. Martinez3, E. Perez de
la Blanca3, M. Ruiz-Galdón4, A. Reyes-Engel4
1
Instituto de Fertilidad Clinica Rincon, I + D Reproduction Unit, Malaga,
Spain
2
Hospital Clínico Universitario Virgen de la Victoria, Análisis Clínicos,
Malaga, Spain
3
Instituto de Fertilidad Clinica Rincon, Reproduction Unit, Malaga, Spain
4
Facultad de Medicina. Universidad de Málaga, Bioquimica y Biología Molecular, Malaga, Spain
Introduction: Folate metabolism disorders have been related with poor fertility outcome, most frequent condition is hyperhomocysteinemia and altered
DNA methylation patterns due to genetics variants and depleted dietary intake
of folate and B12 related vitamins. Periconceptional folic acid and vitamin B12
supplementation has been largely extended during last decade and we have
implemented in recipients undergoing our IVF oocyte donation programs. Genetic condition of recipients has lost interest because of their small contribution
in embryo inheritance. However the association of single nucleotide polymorphisms (SNPs) in the one carbon methyl group pathway and in vitro fertilization (IVF) outcome is still unclear. Our aim is to analyze distribution and association of functional SNPs of enzymes related to methyl group metabolism in
recipients undergoing IVF treatment in our oocyte donation program with fertile tested donors. For this purpose we decided to study distribution of polymorphic markers of key regulating enzyme of Folate and Homocysteine metabolism MTHFR677 (rs1801133), MTHFR1298 (rs1801131), MTR (rs12749581),
CBS (rs5742905) and TCN2776 (rs1801198). Estrogenic metabolism of recipient was assessed for of two relevant SNPs ESR15P (rs2234693) and ESR13P
(rs9340799).
Material and Methods: A total of 42 recipients without child undergoing IVF
treatment in our oocyte donation program were included with average age of
37.8 ( ± 6.5 years) they were women with at least one previous attempt with
own oocyte in IVF treatment. Donors were mothers with children that had provided at least two ongoing pregnancies in no more than 3 embryo transfer in different recipients. Genomic DNA extraction was carried out from buccal swab
(Qiagen Mini KIT). Genotyping of SNPs was developed by PCR multiplex
amplification and minisequencing (SNaPshot™ ABIPRISM 3130). HardyWeinberg equilibrium was assessed for markers in all groups. Chi-squared
test was performed for compare differences between recipients and general
P-559 Young women with good ovarian function and a family record of
monozygotic twins are at a high risk of monozygotic twinning after ART
A. Sobek1, B. Hladikova1, E. Tkadlec2, O. Koutna1, T. Cepelak1, K. Kyselova1,
A.J.R. Sobek1
1
Fertimed, Infertility centre, Olomouc, Czech Republic
2
Palacky university, Dep.of Ecology, Olomouc, Czech Republic
Introduction: There is evidence for at least a 2-fold rise in the incidence of monozygotic twinning (MZT) after assisted reproductive technologies compared
with natural conception. The aim of the study was to evaluate the influence of
micromanipulation techniques (AH, ICSI), length of cultivation, type of cultivation media and the age of patients on the incidence of MZT in IVF/ICSI
patients and to estimate the potential genetic risks for MZT.
Material and Methods: The study group included patients treated for infertility in our IVF centre between 2000–2008. We evaluated the micromanipulation
technique used, length of embryo cultivation, type of cultivation media and the
age of the patient. Ovarian function was determined by basal FSH, the estradiol
(E2) level on the day of oocyte collection, the number of oocytes and consumption of gonadotropins. The parameters of ovarian function in one group of MZT
pregnancies (A) were compared with the population of women with dizygotic
pregnancies (B), single pregnancies (C) and abortions (D). A genetic survey
on multiple pregnancies in the immediate family (up to the third generation)
was done for groups A, B and C. Statistical analysis was done using multiple
regression analysis.
Results: The percentage of monozygotic twins after assisted conception was
1.3% (n = 1535). We found no significant difference between groups A (20), B
(355), C (669) and D (309) in terms of the micromanipulation technique used,
length of embryo cultivation and the type of cultivation media. Patients in group
A were significantly younger 28.6 (A) vs. 29.8 (B), 30.1 (C) and 30.9 (D). Basal
FSH was 7.0 IU/l (A) vs. 7.4 IU/l (B), 7.4 IU/l (C) and 7.7 IU/l (D). E2 level was
3 321 pmol/l (A) vs. 2588 pmol/l (B), 2534 pmol/l (C) and 2575 pmol/l (D).
The number of retrieved oocytes was 16.2 (A), 13.4 (B), 13.0 (C) and 13.1 (D).
The total amount of FSH needed was 2 012 IU (A), 2 217 IU (B), 2222 IU (C)
and 2313 IU (D). The number of embryos transferred was 2.2 (A) vs. 2.8 (B),
2.7 (C) and 2.8 (D). The families of patients in group A had a 41% incidence
of MZT, families in group B had an MZT incidence of 14.1% with a 9.7%
incidence of MZT in family group C (A vs. C p < 0.001).
Conclusions: The incidence of monozygotic twins in women treated for
infertility was 3 times higher than in women conceiving spontaneously.
i331
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Introduction: Previous studies including ours showed the importance of
A-CGH in assessing chromosomal abnormalities in Preimplantaiton Genetic
Screening (PGS) suffering from recurrent IVF failure. The success rate was
encouraging to offer such a screening to any patient with more than 7 recurrent
IVF failures. The current report shows the application of A-CGH on patients
suffering from idiopathic recurrent abortions. Biopsy was performed on embryos at blastocyst stage instead of day 3 stage.
Material and Methods: Patients with 3 consecutive recurrent abortions before
the 9th week of pregnancy were recruited for PGS using A-CGH (agilent platform). Embryos are biopsied day 5 morning and the transfer is performed on
day 6 early morning. The new protocol adopted will be detailed.
Result: Ten patients have been already screened and the result shows abnormalities in many chromosomes. Pregnancy rate is reaching 65%. Detailed results of
the pregnancy duration will be detailed.
Conclusion: This result is the first to use A-CGH with agilent platform on blastocyst biopsy for patients with idiopathic recurrent abortion. Pregnancy rate and
the type of chromosomal abnormalities detected would direct us to highlight
which of the chromosomes to be screened in a less expensive technique like
FISH. More patients will be screened in order to have a more reliable conclusion on the outcome of the protocol adopted.
population frequencies and also between pregnant and non pregnant recipients
condition. Logistic regression models were calculated for odds ratio (OR), 95%
confidence interval (CI), and corresponding p values performed for assess clinical pregnancy outcome and genotype distribution.
Results: Ongoing pregnancy rate were of 52.4%. Distribution of all genotypes
were in HW equilibrium except for markers MTHFR C677T (rs1801133)
(p = 0.049) in non pregnant woman. Higher frequencies of mutant alleles
were found in recipients respect general population in markers MTHFR677
(rs1801133)[X2 = 7,639; p = 0,0219], MTR (rs12749581) )[X2 = 7,639;
p = 0,0219]. However all mutant alleles of the other markers were overrepresented in recipients without statistical significant results. When comparing
clinical pregnancy outcome between recipients, significant differences in genotype frequencies were also found in the MTHFR A1298C (rs1801131) [X2 =
6,615; p = 0,0366] in this case mutant allele was present in higher frequency in
pregnant recipients. The results show that the recipient group is highly selective confirmed by deviation of the genotypes frequencies for the folate gene
polymorphisms studied. Supposedly, due to all women were treated with folate
and Vit B12 supplementation, genetic factor should acquired less relevance. In
fact 2 pregnant recipients presented 3 mutations for the C677T and A1298C
MTHFR polymorphisms (CTCC). This genotype only normally found in fetal
samples, are very rare in general populations (0.002). Strong association of
the A1298C MTHFR mutated allele (OR = 3.72 p = 0.0077 (1.27-10.86) 95%
CI) with the pregnancy outcome could be related with the low incidence of T
allele in the MTHFR C677T SNP considered as functionally more relevant on
ezymatic activity.
Conclusions: Folate gene variants present a high incidence in infertile women
and could be involved in IVF outcome, although further analysis with higher
population need to be completed to extract clinical conclusions. Supported by
Grants SAF2008-03314 and PTQ 09-01-00496.
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
Micromanipulation techniques, the length of cultivation and type of cultivation
media did not influence the incidence of MZT. Ovarian function was markedly
better in women with MZT pregnancies compared to women with dizygotic
and single pregnancies. Women with MZT were significantly younger and had
a lower number of transferred embryos. MZT could be expected after IVF/ICSI
in young women with good ovarian function and with a history of MZT in their
families. These women should be indicated for elective single embryo transfer.
POSTERS:
REPRODUCTIVE SURGERY (FEMALE & MALE)
P. Mora-Guanche1, R. García-Guzmán1, R. Bennett2, L. Iaconianni3,
J. Hernandez1, A. Palumbo1
1
Centro de Asistencia a la Reproducción Humana de Canarias, IVF, La
Laguna, Spain
2
Westchester Reproductive Medicine Mount Kisco, IVF, New York, U.S.A.
3
EcoBi, IVF, Roma, Italy
Introduction: Evaluation of the uterine cavity prior to in vitro fertilization
(IVF) is a key diagnostic step and sonohisterography (SHG) is often used for
this purpose. This study was performed to compare the diagnostic accuracy of
two-dimensional (2D) and three-dimensional (3D) SHG for the diagnosis of
intrauterine lesions in infertility patients.
Materials and Methods: This is a prospective analysis of 209 consecutive 3D
SHG procedures and 70 hysteroscopies performed from January 2009 to December 2009 in a private infertility practice. All patients underwent 3D SHG as
part of the infertility evaluation. All procedures were performed using a Voluson 730 Pro or a Voluson E8 Expert (General Electric Medical Systems, Zipf,
Austria). When an abnormality was present operative histeroscopy was recommended. Data were analyzed comparing 2D and 3D SHG and hysteroscopy
findings.
Results: Of the 209 3D SHGs performed, 110 were normal and 99 abnormal
(47%). Of the abnormals, 70 underwent hysteroscopy. In every one of the abnormal SHGs some intrauterine pathology was found (PPV 100%). Abnormalities
found at 3D SHG included 31 polyps, 10 polyps associated to other pathology, 8
myomas, 27 intrauterine adhesions, 9 uterine septum or subseptum, 12 subseptum associated with other pathology, 1 unicornuate uterus with synechiae and
1 arcuate uterus. The 3D coronal views of the uterus improved the diagnostic
accuracy of the procedure in 64 cases (65% of abnormals, 31% of the total).
A total of 27 patients with abnormal 3D SHG would have been considered
normal by 2D SHG (27/99 = 27%). When only 1 abnormality was present,
the diagnosis made at 3D SHG was confirmed by hysteroscopy in 100% of
cases. When 2 abnormalities were associated, and one of them was a septum
or subseptum, 1 of them was initially missed at 3D SHG in 8 cases. However,
retrospective analysis of the SHG images allowed correct identification and localization of all lesions.
Conclusions: This study demonstrates that 3D SHG has a PPV of 100% for intrauterine lesions. The 3D coronal planes improves the diagnostic of additional
lesions not visible in the 2D images and adds precision to the localization of the
lesions. Experience of the operator is important and there is a learning curve,
but the availability of stored images for later review makes 3D SHG an invaluable tool for the diagnosis of intrauterine pathology.
P-561 Incidence of uterine anomalies in normal and infertile women
and the role of histeroscopic metroplasty in improving the outcome of
fertility treatment
M. Robles Ruiz1, P. Naval Diaz1, R. Garcia-Guzman1, L. Iaconianni2,
J. Hernandez1, A. Palumbo1
1
Centro de Asistencia a la Reproducción Humana de Canarias, IVF, La
Laguna, Spain
i332
Centro Diagnostico Ecografico EcoB.I., IVF, Rome, Italy
Introduction: Congenital uterine malformations have long been associated
with poor obstetrical outcome (miscarriage and premature delivery), but their
role in infertility as been proposed only recently. Three-dimensional ultrasound
(3D-US) has introduced in clinical practice a novel, non-invasive instrument
for the study of uterine morphology that allows evaluation of the incidence of
uterine anomalies in both normal and infertile women. This study was designed
(1) to detect the incidence of uterine anomalies in an infertility population in
comparison with fertile women and (2) to assess the impact of hysteroscopic
metroplasty on the outcome of fertility treatment.
Material and Methods: A retrospective review of our electronic medical records (EMR) (Viewpoint, GE, Zipf, Austria) evaluating all 3D-US studies performed from July 2008 to June 2009 was performed. 583 consecutive women
seen in a single private gynaecologic center either for infertility (n = 458, 79%)
or for routine gynecologic care (n = 125, 21%) were included. US were performed using a Voluson 730 or a Voluson E8 expert; the 3D volumes of the
uterus and patients´ medical histories stored in our EMR, were retrieved and the
4D images reviewed using the software 4D view (GE, Zipf, Austria). The diagnosis of uterine malformations was made by 3D-US in all cases and confirmed
by 3D sonohisterography in 43 cases; 39 patients underwent hysteroscopy and
histeroscopic metroplasty as appropriate.
Results: The uterine morphology was normal in 489 women (83.9%) while
94 (16.1%) has some uterine anomaly: of these 79 (84%) were found in infertile patients and 15 (16%) in routine gynaecologic patients. Of the 94 uterine
anomalies 42 were subseptate uteri (45%), 41 were arcuate uteri (44%), 8 were
septate uteri (8%), 2 were unicornuate (2%), 1 bicornuate uteri (1%). Of the 125
routine gynaecologic cases 15 (12%) had a uterine anomaly; 13 were arcuate
uteri (10.4% of the total) and 2 were subseptate uteri (1.6%). Of the 458 infertile women 79 (17%) had a uterine anomaly; 28 were arcuate uteri (6.1%), 40
subseptate (8.7%) and 8 septate (1.7%), 2 unicornuate (0.4%) and 1 bicornuate
(0.2%). Hysteroscopic metroplasty was performed in 39 cases, all with infertility (2 arcuate uteri, 32 subseptate uteri and 5 septate uteri) and subsequent IVF
treatment in these cases resulted in a pregnancy rate of 58%, as opposed to a
PR of 24% in the group of patients in which the uterine anomaly was not corrected. Patients in the routine gynaecologic group had no history of miscarriage
or infertility. Of the 79 infertile women diagnosed with a uterine anomaly 21
(26.6%;17 subseptate uteri and 4 in arcuate uteri) had a history of one or more
miscarriages.
Conclusions: This study shows that the incidence of uterine anomalies is significantly higher in patients with infertility, than in normal women, suggesting
a role of uterine malformations in the pathogenesis of infertility. Based on these
findings, hysteroscopic metroplasty may be indicated in these cases even in the
absence of a history of recurrent pregnancy loss. The increased pregnancy rates
reported after surgical correction of the uterine malformation further supports
this conclusion.
P-562 Recovery rates and treatment outcome after testicular biopsy for
male factor infertility
I. Afaneh1, R. Roopnarinesingh1, E. Mocanu1
1
HARI Unit, Rotunda Hospital, Dublin, Ireland
Introduction: Severe male factor infertility and in particular azoospermia is a
diagnosis made with increased frequency in today’s clinical ART practice. We
aimed to review the outcome of testicular biopsies and subsequent ICSI treatment outcomes in couples with the diagnosis of severe male factor.
Methods: This was a 2-year retrospective individual chart review of all cases
where a testicular biopsy was performed at the Human Assisted Reproduction Ireland (HARI) Unit, a tertiary referral academic centre. The following
variables were recorded: age of partners, male/female FSH, type of infertility,
underlying cause of male infertility, male genetic profile, rate of freeze after biopsy, parameters of extracted sperm, type of stimulation protocol, number and
grade of embryos, day of transfer and pregnancy outcome. Data was analysed
using SPSS.
Results: We identified 49 cases of testicular biopsy (TB). Primary infertility
was diagnosed in 33(65%) couples. The indication for TB was azoospermia in
44(90%) of cases while the rest had extreme oligozoospermia requiring biopsy.
The cause of azoospermia was obstructive in 31(63%) men and non-obstructive
in 17 (27%). With the exception of failed reversal of vasectomy, genetic profile was performed in all other cases. All had normal karyotype 46XY, 2 were
Downloaded from https://academic.oup.com/humrep/article-abstract/25/suppl_1/i321/590818 by guest on 20 June 2020
P-560 Diagnostic accuracy of three dimensional sonohysterography
(SHG) for intrauterine abnormalities in infertility compared to two
dimensional SHG
2
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