SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - 174A

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Reproductive Sciences Vol. 25, Supplement 1, March 2018

METHODS: To detect possible changes in mtDNA concentrations, a
new mtDNA quantitative analysis in CGCs was designed using qPCR,
constructed primers were completely validated for quantitative analysis.
HepG2 cell cultures treated with EtBr showed a decrease in mtDNA
of more than half compared to untreated controls. CGCs were isolated
by mechanical separation at the time of oocyte retrieval from patients
undergoing standard ovarian stimulation for in-vitro fertilization. Pools
of CGCs were then plated, cultured, and treated with MPB (6, 300, 657,
1000 nM), BPB (2, 5.1, 20, 1000 nM), and BPA (1.4, 8.7, 500, 1000
nM) for 24 h. Total DNA extraction was performed using the All-In-One
DNA/RNA Miniprep Kit from Bio Basic (BS88203, Ontario, Canada).
Differences between groups were determined by one-way ANOVA with
a post hoc Dunn´s test. P-values <0.05 (two-tailed) were considered
statistically significant.
RESULTS: HepG2 cell cultures treated with EtBr showed a decrease
in mtDNA of more than half compared to untreated controls. MtDNA
content in treated cultures was compared with untreated controls. There
was no significant effect of MPB on CGCs' mtDNA content, nevertheless,
a significant decrease was observed in CGCs treated with BPB at 1000
nM (around 27 %) this goes in accordance to the fact that the toxicity
of parabens increases with the length of the alkyl group. Similarly, a
decrease in mtDNA content was also observed in cultures treated with
environmentally relevant concentrations of BPA (8.7, 500 and 1000 nM;
27 to 39%, p< 0.05), and with other really high concentrations of BPA
reported recently (4.3 and 43 μM). In all cases cell viability was unaltered.
Also, Diethylstilbestrol, a potent synthetic endocrine disruptor, caused a
~40% decrease in mtDNA.
CONCLUSION: We concluded that BPB and BPA cause mitochondrial
dysfunction at mtDNA level suggesting that mtDNA is an important intramitochondrial target of these endocrine disruptors.

T-195
A Predictive Fertility Treatment Model Based on Oocyte Quality and
Reactive Oxygen Species. Charalampos Chatzicharalampous†, Roohi
Jeelani*, Robert Morris*, Sana Khan*, Husam Abu-Soud*. Wayne State
University, Detroit, MI, United States.
INTRODUCTION: Myeloperoxidase (MPO) is an abundant hemecontaining enzyme, known to generate reactive oxygen species (ROS)
such as hypochlorous acid from hydrogen peroxide and chloride. It is
produced in high levels during inflammation and is associated with poor
reproductive outcomes. MPO is also present in neutrophils, monocytes,
and macrophages. Previous research has shown high levels of these
immune cells in the peritoneal cavity of patients with pelvic inflammatory
disease, endometriosis and polycystic ovarian syndrome. There is a paucity
of information regarding the mechanism of damage in response to ROS;
however, an initial step appears to be the disassembly of the microtubule
organizing center (MTOC), which is part of the cellular scaffold that
mediates intracellular movement. One of the key proteins in the MTOC,
pericentrin, located at the ends of the spindle structure, can be used as
a marker of oocyte quality. In normal oocytes, pericentrin is primarily
assembled as a condensation at both spindle poles, by utilizing the
acentrosomal MTOC. The spindle has a symmetrical pointed barrel shape,
assembled around the chromosomal plate at the spindle equator. Here we
investigate the mechanism of activated immune cells in the deterioration
of oocyte quality through disruption of the spindle and its protection by
melatonin, an MPO inhibitor and ROS scavenger.
METHODS: Mouse metaphase II oocytes with and without cumulus
cells (n=200/200) were co-cultured in human tubular fluid (HTF) media,
at 37°C, with activated mouse peritoneal macrophages and neutrophils
(1.0 x 106 cells/ml) in the absence and presence of melatonin (200 μM),
for different incubation periods (3, 6 and 12 hr). Oocytes were then fixed,
stained and their quality was scored based on alterations in the pericentrin
localization, microtubule morphology and chromosomal alignment.
RESULTS: Oocytes were negatively affected when co-cultured with
activated mouse peritoneal macrophages and neutrophils. Evidence of
deteriorating oocyte quality was observed as distortion of the spindle
and chromosomal material and de-localization of pericentrin proteins in
a time dependent fashion as compared to controls. At longer incubation

Scientific Abstracts

times, the spindle fibers were bent or unevenly broken, and the MTOC
proteins were further dispersed or undetectable. In all cases the presence
of cumulus cells offered no protection; however significant protection
was offered in the group of oocytes treated with melatonin.
CONCLUSION: This work provides a direct physiologic link between
peritoneal inflammation and decreased oocyte quality. Movement or
destruction of pericentrin represents dysregulation of the MTOC which is
associated with decreased oocyte quality and may serve as a biomarker for
functional competence. Melatonin, acting as an MPO inhibitor and ROS
scavenger can be helpful in protecting oocytes from adverse outcomes.

T-196
Day 3 Embryo Scoring: Can We Accurately Predict "Biopsiable"
Blastocysts? Stephanie R Baum†,1 Moti Gulersen†,1 Avner Hershlag,2
Liron Bar-El,2 Christine Mullin,2 Sara Bristow,2 Matthew Cohen,2 Tomer
Singer.1 1Lenox Hill Hospital, Northwell Health, New York, NY, United
States; 2Northwell Health, New York, NY, United States.
INTRODUCTION: Preimplantation Genetic Screening (PGS) is an
IVF procedure to examine embryos for chromosomal abnormalities. This
technique is used to identify euploid embryos that should be selected
for transfer.
Our study aims to demonstrate that morphological criteria of a Day 3
embryo can be used to determine which embryos will ultimately develop
to a "biopsiable" blastocyst to undergo preimplantation genetic screening.
METHODS: We reviewed 89 patients undergoing in vitro fertilization
(IVF) with intracytoplasmic sperm insemination (ICSI) and PGS in a
single academic institution from January-October 2016. We excluded
patients with azospermia, patients >45, and patients with no embryo
development beyond Day 2. Included embryos were divided into 2 groups:
biopsied blastocysts and discarded Day 6 embryos. Each embryo received
a composite score based on Simplified cleavage stage SART embryo
scoring system. Mean composite scores for each cohort were calculated.
Other outcome variables included maternal age, ovarian reserve measures,
and cycle outcome. The composite score includes 4 variables: # of cells,
appearance, homogeneity of cells and degree of fragmentation. The sum
of the 4 variables is the composite score. # of cells: 8 cells = 4 points, 7
or 9 cells = 3, 6 or 10 cells = 2 and 5 or less cells or 11 or greater cells
= 1. Appearance scoring is as follows: good = 3 points, fair = 2, and
poor = 1. Homogeneity of cells scoring is as follows: "A" (uniform cell
size) = 3 points, "B" = 2, "C" = 1. Degree of fragmentation scoring:
"A" (<20%) = 1 point, "B" (20-50%) = 2 and "C" (>50%) = 3. The best
possible composite score is 13 (8GAA). The worst possible score is 4.
We performed a 2-sample t-test assuming unequal variance. This study
is a retrospective analysis.
RESULTS: The embryos that underwent biopsy had a statistically
significant higher (better) composite score on Day 3 than those that were
discarded: 10.9 +/- 1.8 versus 9.4 +/- 2.3, respectively. Over 56% of
embryos with a composite score of 11 or greater were biopsied whereas
more than 90% of embryos with a score of 6 or less were discarded.
Baseline and Group Characteristics (data represented as Mean +/- SD)
Discarded (No
biopsy)

Blastocyst
(Biopsy)

p-value
(T-test)

Number of Embryos

440

330

Maternal Age

37.8 +/- 4.9

36.2 +/- 5.0

P<0.001

Maternal AMH

6.3 +/-5.3

5.5 +/- 4.9

P<0.05

Maternal FSH

6.8 +/- 2.0

6.9 +/- 1.9

NS

Number of retrieved
oocytes/cycle

23.1 +/- 12.6

21.7 +/- 11.8

NS

Number of mature M2
oocytes/cycle

18.5 +/- 10.8

17.2 +/- 9.6

NS

Number of 2PN/cycle

15.4 +/- 9.5

13.7 +/- 8.0

P<0.05

Morphological score

9.4 +/- 2.3

10.9 +/- 1.8

P<0.001

CONCLUSION: By assigning specific morphological criteria in the
form of a grade on Day 3 of embryo development, we were able to show
that a higher composite score (higher quality) is correlated with biopsied



Table of Contents for the Digital Edition of SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018

SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - Cover1
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SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - Cover3
SRI Supplement to Reproductive Sciences - Volume 25 Number 1 - March 2018 - Cover4
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2020
https://www.nxtbook.com/nxtbooks/sage/psychologicalscience_demo
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2020
https://www.nxtbook.com/nxtbooks/sage/fai_202009
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_august2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2020
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2019
https://www.nxtbook.com/nxtbooks/sage/fai_201909
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_july2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2019
https://www.nxtbook.com/nxtbooks/sage/canadianpharmacistsjournal_05062019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2019
https://www.nxtbook.com/nxtbooks/sage/sri_supplement_201903
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2019
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2018
https://www.nxtbook.com/nxtbooks/sage/tec_20180810
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_julyaugust2018
https://www.nxtbook.com/nxtbooks/sage/fai_201807
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_april2018
https://www.nxtbook.com/nxtbooks/sage/sri_supplement_201803
https://www.nxtbook.com/nxtbooks/sage/slas_discovery_201712
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_february2018
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_december2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_november2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_october2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_september2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_julyaugust2017
https://www.nxtbook.com/nxtbooks/sage/fai_supplement_201709
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_june2017
https://www.nxtbook.com/nxtbooks/sage/hospitalpharmacy_may2017
https://www.nxtbook.com/nxtbooks/sage/fai_201706
https://www.nxtbook.com/nxtbooks/sage/fai_201607
https://www.nxtbookmedia.com