Document Type : Original Article
Authors
1 Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, Lebanon
2 Al-Hadi Laboratory and Medical Center, Beirut, Lebanon
3 Lebanese University, Faculty of Sciences, Section II, Fanar, Lebanon and Azoury IVF Clinic, Mount Lebanon Hospital, Camille Chamoun Bvd, Beirut, Lebanon
4 4OB-GYN Department, Inova Fairfax Hospital, Falls Church, Virginia
Abstract
Keywords
Granulosa cells (GCs) are the somatic cells surrounding the oocyte in the ovary (
In assisted reproductive technology, GCs can be collected from follicular fluid (FF) during oocyte retrieval,
form women undergoing controlled ovarian stimulation
(COS) (
The efficiency of purification methods that are based
on the differential physical properties of LGCs and contaminting cells were tested in several reports (
Therefore, the aim of present study was to test whether isolating granulosa Aggs at the beginning of purification procedure would decrease the percentage of contaminating cells at the DG interface. In order to answer this biological question, we collected the granulosa Aggs (which are larger than other FF contaminants) directly from the FF and then subjected them to the DG centrifugation. Next, we compared the outcome of this modified protocol to that of traditional one. This comparison was performed in terms of the percentage of recovered LGC, vitality and purity.
FFs were collected from preovulatory follicles of
young women (<38 years old) undergoing oocytes retrieval for intra-cytoplasmic sperm injection (ICSI),
via transvaginal ultrasound-guided aspiration (n=32)
(
COC-free FFs (n=32) were randomly assigned to one
of two IVF GC preparation methods. The first technique
was positive selection of granulosa Aggs, after DG centrifugation (DG): DG/Agg (
Each FF was pooled into a 14 ml falcon tube and centrifuged for 10 minutes at 2000 rpm. The collected pellet
was gently pipetted onto a DG made up of two layers:
40% and 80% (Sperm Gradient Kit, Sydney IVF, COOK
medical, EMEC Lebanon). After centrifugation for 10
minutes at 1200 rpm, the ring-like layer at the interface
was transferred into a 60 mm petri dish. The Aggs were
positively selected under a dissecting microscope and
washed in human tubal fluid medium (HTF medium, Life
Global, Ibra Haddad Lebanon). The wash consisted of a
centrifugation for 10 minutes at 2000 rpm. Next, the pellet was resuspended in 1 ml HTF. Then, Aggs breaking
up was performed mechanically, using a Pasteur pipette
(
Aggregates were collected from the COC-free FF, in
HTF medium. Next, these Aggs were gently pipetted
onto a DG made up of two layers: 40% and 80%. They
were next centrifuged for 10 minutes at 1200 rpm.
The ring-like layer in the interface was then transferred into a 5 ml round tube, mixed with 1 ml HTF
and centrifuged for 10 minutes at 2000 rpm. At the
end, the pellet was suspended in 1 ml HTF followed
by up- and down-pipetting for 1 minute, to dissociate
the Aggs (
The purified GCs were counted using a hemocytometer
microscopic slide and cell viability was determined using
Trypan Blue (0.4%) (
In order to compare purity of the cell suspension and the
LGCs morphology derived from DG/Agg and Agg/DG
techniques, a thin smear slide was prepared from each cell
suspension (
The schematic of luteal GCs purification procedures. A. Follicular fluid samples (n=16) were subjected to a simple wash, then subjected to a two layered (40-80%) density gradient centrifugation. After that, the luteal Aggs were transferred into a petri dish using a micropipette, washed and dissociated. Finally, LGC count, purity, vitality and morphology were assessed. B. Using a micropipette, luteal Aggs were collected from the FF samples (n=16), moved into washing drops to reduce contamination and then subjected to a two layered (40-80%) density gradient centrifugation. After that, the Aggs were washed, dissociated and analysed in terms of LGC count, purity, vitality and morphology. GCs; Granulosa cells, Aggs; Granulosa cell aggregates, FF; Follicular fluids, LGCs; Luteal GCs, and DG; Density gradient.
A cluster of granulosa cells stained with Wright stain. A cluster of granulosa cells (GCs) stained with Wright stain. The GC is made up of a central nucleus (purple) and foamy cytoplasm (clear purple): solid arrows. A white blood cell (WBC, multi-lobed nucleus) was observed: dashed arrow (magnification: ×100 under immersion oil).
Statistical analysis was performed using IBM SPSS 23 software (IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp). Data with normal distribution were then compared using independent samples t test. Data with non-normal distribution were compared using the Mann-Whitney non-parametric test. All data were presented as median [interquartile range (IQR)]. Categorical data were compared using Chi-square test. Results were considered statistically significant for a P<0.05.
There was no statistically significant difference in the
female age (P=0.5), number of retrieved oocytes (P=0.2)
and infertility etiology (P=0.8) between these two techniques (
Comparison of the population characteristics in DG/Agg and Agg/DG groups
Population characteristics | DG/Agg technique | Agg/DG technique | P value | |
---|---|---|---|---|
Infertility etiology | Female age (Y) | 32.25 ± 5.4 | 31.06 ± 5.35 | 0.5 |
Number of retrieved oocytes | 9 ± 6.23 | 12 ± 6.10 | 0.2 | |
Male factor (%) | 6/16 (37.5) | 5/16 (31.3) | 0.8 | |
Female factor (%) | 4/16 (25) | 6/16 (37.5) | ||
Male and female factors (%) | 2/16 (12.5) | 1/16 (6.3) | ||
Unexplained infertility (%) | 4/16 (25) | 4/16 (25) | ||
Data are presented as mean ± SD or n (%).
Results are expressed as mean ± standard deviation (SD) for normally distributed continuous variables and percentage for categorical data. Continuous variables were compared using the independent samples t test. Categorical data were compared using the Chi-square statistical test. There were no statistically significant difference between the groups of female age (P=0.5), number of retrieved oocytes (P=0.2) and infertility etiology (P=0.8). P>0.05 indicates that there is no statistically significant difference between two groups.
Hemocytometer slide and trypan blue staining were
used to assess total cell concentration and vitality after
two purification techniques. In one hand, a significantly
lower concentration of cells was obtained after Agg/DG
compared to DG/Agg (P<0.001,
A thin smear was prepared from each cell suspension,
after processing and they were stained using WrightGiemsa stain (
Boxplots show the total cell concentration and vitality percentage in DG/Agg and Agg/DG techniques. Boxes indicate the interquartile range. Horizontal bars within the boxes indicate the median. Whiskers indicate the range of data. Data were compared using the Mann-Whitney non-parametric test. A. Boxplot shows a lower concentration of all cell types after Agg/DG technique compared to DG/Agg technique (***; P<0.001). B. Boxplots show no statistically significant difference in the vitality percentage of cells after two techniques. NS; No significant difference.
Boxplots show total recovered granulosa and percentage of GCs with normal morphology in DG/Agg and Agg/DG techniques. Boxes indicate the interquartile range. Horizontal bars within boxes indicate the median. Whiskers indicate the range of data. Data were compared using the Mann-Whitney non-parametric test. A. Boxplot shows a higher percentage of total GCs after Agg/DG compared to DG/Agg (***; P<0.01), B. Boxplots show that there was no statistically significant difference in the total granulosa count after DG/Agg and Agg/DG techniques. C. Boxplots show a higher percentage of GCs with normal morphology in the Agg/ DG group companed to the DG/Agg group. NS; No significant difference.
Purity of each preparation was estimated on WrightGiemsa stained smears (
Boxplots show the percentage of contaminant cell types presented after DG/Agg and Agg/DG techniques. Boxes indicate the interquartile range. Horizontal bars within boxes indicate the median. Whiskers indi- cate the range of data. Data were compared using the Mann-Whitney non-parametric test. A. Boxplots show a lower percentage of white blood cells in the Agg/DG technique compared to the DG/Agg technique (**; P<0.01). B. Boxplots show a lower percentage of red blood cells in the Agg/DG technique compared to DG/Agg technique (***; P<0.001). C. Box- plots show a lower percentage of epithelial cells in the Agg/DG technique compared to the DG/Agg technique (**; P<0.01).
The aim of present report was to decrease percentages of contaminating cells in the suspension obtained from the positive selection of granulosa Aggs after DG procedure. Here we showed that collecting Aggs from the FF prior to DG centrifugation significantly decreased the percentages of contaminating cells.
In assisted reproductive technology, aspirated human
FF contains heterogeneous population of cells (
Studying quality, quantity and gene expression of the
GCs may improve the information given about ovarian
function and oocyte physiology (
The purification strategies that are based on the immunorecognition of specific cell markers, such as fluorescence activated cell sorting (FACS), magnetic activating
cell sorting (MACS), and Dynabeads, are considered to
be the most efficient in terms of purity and less efficient in
LGCs recovery (
Interestingly, our proposed protocol (positive selection
of granulosa Aggs before DG centrifugation) led to a lower contamination by red blood cells, white blood cells and
epithelial cells. Actually isolating granulosa Aggs from
the heterogeneous cell population at the beginning of the
purification procedure could explain the lower level of
contamination obtained using this modified protocol. It is
essential to mention that application of a cell strainer to
collect GCs can also reduce the level of white blood cells
despite being more expensive than our proposed procedure (
In addition, our modified protocol resulted in a vitality percentage as high as the original procedure (positive
selection of granulose Aggs after density gradient). Strikingly however, the percentage of granulosa with normal
morphology was higher in the suspension obtained from
our modified procedure compared to the original procedure. In fact, GCs are very sensitive to reactive oxygen
species (ROS) (
The positive selection of GCs before subjecting them
to a DG centrifugation surpassed the original procedure
in terms of purity and recovery of granulosa with normal
morphology. It resulted in a relatively high number of recovered LGCs less contaminated by other cell types. In
addition to its efficiency, the modified protocol is simple,
inexpensive and rapidly operated. That being the case, it
could be a method of choice to prepare GCs for research
purposes in clinical settings