Keywords
single embryo culture - cf-DNA - spent media - noninvasive PGT - blastocysts
Introduction
Infertility presents a significant challenge in modern medicine, recognized by the
World Health Organization as a societal issue due to its widespread impact. In addressing
infertility, various treatment approaches exist, including medication-based therapy,
surgical interventions, and Assisted Reproductive Technology (ART).[1] ART has seen remarkable advancements since the birth of Louise Brown in 1978, with
subsequent successes globally.[2]
However, challenges persist, notably in genetic screening of embryos, where accurate
detection of genetic defects is crucial for successful pregnancy outcomes.[3] Current techniques such as Preimplantation Genetic Testing (PGT) for Aneuploidy
have shown effectiveness but raise concerns about embryo safety and procedural standardization.
The emergence of noninvasive Preimplantation Genetic Testing (niPGT) offers a promising
alternative, potentially reducing costs and improving outcomes.[3] Studies have shown niPGT's reliability in detecting genetic anomalies, with less
risk of error than invasive techniques.
Innovations in ART also include noninvasive embryo monitoring through spent media
analysis, which minimizes embryo manipulation and potential harm due to biopsy.[4]
[5]
[6] Additionally, research into group embryo culture highlights the importance of autocrine
and paracrine communication in promoting embryonic development.[7] Despite advancements, questions remain regarding the suitability of single-drop
culture for embryo development and also for getting sufficient sample volume to perform
further procedures.
Furthermore, PGT has significant applications not only in human medicine but also
in animal husbandry. In domestic animals, PGT is utilized to introduce desirable traits,
enhance genetic diversity, and improve overall herd quality.[8]
[9]
[10] By selecting embryos with specific genetic characteristics, breeders can promote
traits such as disease resistance, productivity, and longevity, contributing to the
advancement of livestock breeding programs.[11]
[12] Selection of the embryos based on genomic evaluation would increase selection intensity
and reduce generation interval leading to increased genetic gain. The comparison between
single and group culture methods is essential to establish a process that maintains
embryo production efficiency while providing noninvasive insights into the blastocysts.
If single culture does not achieve embryo production efficiency comparable to group
culture, it would not be adopted by laboratories focused primarily on producing embryos.
Ensuring similar efficiency is crucial for the acceptance of this method for noninvasive
embryo testing.
Materials and Methods
Materials
The study was conducted at the OPU-IVEP-ET laboratory of the National Dairy Development
Board (NDDB), Anand. While bovine oocytes were sourced from NDDB, Anand, and frozen
semen doses (FSDs) were obtained from Sabarmati Ashram Gaushala, Bidaj. Oocyte washing
media, in vitro maturation (IVM), in vitro fertilization (IVF), in vitro culture (IVC)
media, semen washing media, and density gradient media were sourced from VitroGen,
Brazil. Standard equipment for embryo culture was utilized. DNA extraction was conducted
using the QIAamp Circulating Nucleic Acid Kit from QIAGEN (catalog number/ID: 55114,
Germany), while quantitative measurement of cf-DNA was performed using the Qubit 4
Fluorometer (Brand: Invitrogen™ Q33226, Fisher Scientific, Sweden) with the Qubit
1× dsDNA HS Assay Kit (catalog number: Q32851, Thermo Fisher Scientific, MA, United
States).
Methods
Sample Size and Cycle Number
A total of 14 ovum pickup (OPU) and in vitro embryo production cycles (OPU-IVEP) were
conducted and a total of 239 oocytes were aspirated. Seven cycles were each subjected
to single embryo culture and group embryo culture, respectively. Spent media were
collected from each cycle involving single-drop embryo culture plates for DNA extraction
and noninvasive Preimplantation Genetic Diagnosis protocol standardization.
The in vitro embryo production (IVEP) was carried out as per already standardized
procedures in the laboratory with some minor modifications.[13]
[14]
[15]
[16] Brief details of the process are mentioned below in subsections “Ovum Pickup” to
“In Vitro Culture.”
The grading of cumulus–oocyte complex (COC) and embryos was performed as per standards
prescribed by the International Embryo Technology Society (IETS) in the Manual of
the IETS, 5th Edition, to keep uniformity with the internationally followed norms.
Ovum Pickup
The OPU procedures were performed according to the standardized procedure in the laboratory
for Transvaginal Ultrasound-guided OPU in cattle. Oocytes were aspirated using OPU
media. All visible follicles were counted and aspirated using an OPU needle coupled
to the aspiration line and a vacuum system.
In Vitro Maturation
The oocytes were searched from the aspirated solution containing the COCs. Collected
oocytes were graded according to IETS guidelines before processing for IVM. The grading
was based on cumulus density and characteristics of ooplasm, viz., Grade 1 included
COCs with more than three complete and compact layers of cumulus cells, Grade 2 had
one or two compact cumulus cell layers, Grade 3 had less than one complete layer,
and Grade 4 had expanded cumulus cell layers or signs of degeneration ([Fig. 1]). The good-quality oocytes (Grades 1–3) were washed in IVM media and graded before
placing them in preequilibrated IVM drop (media were preequilibrated overnight). Oocytes
with maturation media were placed in an incubator with 5% CO2 in air (O2 concentration around 14–16%), 38.5 °C temperature, more than 90% relative humidity
(RH), and incubated for around 22 hours for IVM.
Fig. 1 The figure illustrates various grading patterns of cumulus–oocyte complexes as outlined
in the IETS Manual, 5th Edition. Grade 1: COCs have more than three complete and compact layers of cumulus cells covering
the surface of the zona pellucida (ZP). The ooplasm is dense and has even granulation.
Grade 2: COCs have one or two compact cumulus cell layers covering the surface of the zona
pellucida. Grade 3: COCs have less than one complete layer of cumulus cells covering the surface of the
ZP. COC, cumulus–oocyte complex; IETS, International Embryo Technology Society.
In Vitro Fertilization
Oocyte Transfer
Oocytes were washed with IVF media and then transferred to preequilibrated IVF media
drops (media were preequilibrated overnight) after approximately 22 hours of IVM.
Sperm Preparation and Fertilization
Preselected FSDs were used for fertilization. The semen underwent a two-step centrifugation
process. Initially, the semen layered over the upper and lower gradient layers was
centrifuged at 2,000 × g for 5 minutes. The pellet was then resuspended in IVF media
and centrifuged again at 500 × g for 5 minutes. Finally, a predetermined volume of
the sperm suspension was added to each fertilization drop. The Petri dish was then
placed in an incubator with 5% CO2 in air (O2 concentration around 14–16%), 38.5 °C temperature, more than 90% RH, and incubated
for around 18 hours for IVF ([Fig. 2]).
In Vitro Culture
The presumptive zygotes (fertilized oocytes) underwent a denuding process following
fertilization. This step involved carefully removing all surrounding cumulus cells
using a denuding pipette. The denuding process was performed slowly and with utmost
care to prevent damage to the zona pellucida. After complete denuding, the presumptive
zygotes were washed sequentially in wash media and an IVC medium. Finally, the washed
presumptive zygotes were transferred to preequilibrated IVC media drops (media were
preequilibrated overnight) and placed back in a Mixed Gas benchtop incubator with
5% CO2, 5% O2, 90% N2, 38.5 °C temperature, more than 90% RH for 7 days from the date of IVF.
Single-Drop Embryo Culture and Grading of Embryos
For single embryo culture, individual Day 3-grown embryos were transferred to separate
single-drop embryo culture plates, where they were allowed to continue growing for
the remaining days until Day 7. After 7 days from the IVF, the embryos were graded
as per IETS guidelines, as outlined in the Manual of the IETS, 5th Edition. Embryos
are assigned numerical codes from 1 to 9 based on their developmental stage, ranging
from unfertilized or one-celled embryos on day 1 to expanding hatched blastocysts
on days 9 and 10. For the current study, only Expanded blastocysts (day 7) and Blastocysts
(day 7) were considered. Additionally, embryos undergo grading based on their quality,
with numerical codes indicating their morphological integrity. Grade 1 represents
excellent or good embryos, Grade 2 denotes fair quality, Grade 3 signifies poor quality,
and Grade 4 indicates dead or degenerating embryos ([Fig. 3]).
Spent Media Collection Protocol
On day 7 from IVF, blastocysts were graded, and spent media from single-drop cultures
were collected. Spent media were labeled and stored for further analysis or immediate
DNA extraction.
Isolation of Cell-free DNA
cf-DNA was extracted from spent media using the QIAamp Circulating Nucleic Acid Kit
according to the manufacturer's instructions. The extraction process involved various
steps including lysate preparation, column purification, and elution.
Quantification of Cell-free DNA
Quantification of cfDNA was performed using the Qubit fluorometer 4.0 in combination
with the Qubit 1× dsDNA HS Assay Kit. Samples were diluted in Qubit working solution
before quantification as per the manufacturer's instructions.
Results
Phase 1: Embryo Culture Efficiency
Comparison of Group versus Single Embryo Culture
A comparative analysis between group embryo culture and single-drop embryo culture
methods revealed comparable trends in embryo formation efficiency across various cycles.
Both methods exhibited variability in blastocyst rates, with single-drop embryo culture
demonstrating a higher average blastocyst rate (40.6%) compared to group embryo culture
(27.5%). However, statistical analysis indicated no significant difference between
the two culture methods (p = 0.09). The findings suggest that while single-drop embryo culture may offer slightly
higher efficiency, both methods are suitable for supporting blastocyst development
in IVEP cycles ([Table 1]; [Figs. 1]
[2]
[3]
[4]).
Fig. 2 Oocyte with cumulus and sperm suspended in the media for fertilization (seen under
an inverted microscope at 40 × ). (A) Sperm is suspended in the media for fertilization.
(B) Cumulus cells. (C) Ooplasm. (D) Zona pellucida.
Fig. 3 The figure presents the grading patterns of embryos on days 7 and 8 in accordance
with the IETS Manual, 5th Edition. Grade 6: Blastocyst (days 7–8). Grade 7: Expanded blastocyst (days 8–9). Grade 8: Hatched blastocyst (day 9). Grade 9: Expanding hatched blastocyst (days 9–10). IETS, International Embryo Technology Society.
Fig. 4 Biostatistics graph with an error bar of group embryo culture versus single embryo
culture. The conversion rate of group embryo culture versus single embryo culture;
data are expressed as mean ± SEM (n = 7) 13.58 ± 7.410 when compared between the group embryo culture samples and single
embryo culture (p-value = 0.09), which shows there is no significant difference between the two culture
groups; therefore, the Null Hypothesis is accepted.
Table 1
Comparison of group embryo culture versus single embryo culture
|
Cycle number
|
Group embryo culture
|
Single-drop embryo culture
|
|
Number of oocytes kept in in vitro culture
|
Number of embryos formed
|
Conversion rate
|
Number of oocytes kept in in vitro culture
|
Number of embryos formed
|
Conversion rate
|
|
1
|
10
|
3
|
30%
|
8
|
2
|
25%
|
|
2
|
24
|
8
|
33%
|
12
|
4
|
33.33%
|
|
3
|
6
|
1
|
17%
|
25
|
10
|
40%
|
|
4
|
13
|
5
|
38.46%
|
9
|
7
|
77.78%
|
|
5
|
25
|
9
|
36%
|
25
|
10
|
40%
|
|
6
|
17
|
3
|
17.65%
|
17
|
6
|
35.29%
|
|
7
|
24
|
5
|
20.83%
|
24
|
8
|
33%
|
Phase 2: Cell free-DNA Release by Single Embryos
In the second phase, the release of cell-free DNA (cf-DNA) by single embryos into
culture media was investigated. A total of 48 samples were analyzed across seven cycles.
The mean quantity of cf-DNA released by single embryos was found to be 365 ± 10.25
pg/μL. Statistical analysis revealed a significant difference in cf-DNA release between
samples (p < 0.0001), validating the potential use of DNA fragments retrieved from spent media
for noninvasive genetic analysis ([Fig. 5]).
Fig. 5 Biostatistics graph with an error bar of total cf-DNA released by a single embryo
to culture media. Total cf-DNA released by a single embryo to culture media (graph
a); data are expressed as mean ± SEM (n = 48) p < 0.0001, 365 ± 10.25 between the samples. As there is a significant difference,
the Null hypothesis is rejected and it validates that DNA fragments retrieved from
spent media can be used for the noninvasive method. cf-DNA, cell-free DNA.
[Figures 3] and [5] visually represent the data and statistical analyses conducted in this study, providing
insights into the efficiency of embryo culture methods and the release of cf-DNA by
single embryos.
Discussion
Developing a noninvasive method for the evaluation of genetic insight into an embryo
is always desirable considering the damaging effects of the biopsy process. Further,
zona-damaged embryos are not preferred for the international trade of bovine embryos
considering the potential to transmit diseases between boundaries. Thus, it is imperative
to develop and optimize a single embryo culture method for developing an alternative,
and without a single embryo culture, it is not possible to get insight into a particular
embryo. The comparable result of single embryo culture compared to group culture may
be explained by the fact that the sole nutrition from the culture media is devoted
to a single embryo.[17] Aneuploid embryos in IVF, which can range from 20 to 100%, present significant biological
challenges.[18] These chromosomal abnormalities commonly result from errors in chromosome segregation
during meiosis,[19] influenced by factors such as advanced parental ages, eating habits, and lifestyle.
Such aberrations significantly contribute to early pregnancy miscarriages and severe
chromosomal disorders. Research indicates that the outcomes of ART procedures can
be improved through PGT for aneuploidy, which facilitates the selection of embryos
with optimal chromosomal integrity.[19]
The global use of PGT is on the rise, typically involving invasive methods like polar
body, blastomere, trophectoderm biopsy, or blastocentesis, to extract embryonic DNA.[20] However, these methods come with technical constraints and ethical considerations.
Two primary issues spark controversy within the scientific community: the need for
specialized instrumentation and skilled practitioners to ensure embryo viability,
and concerns about potential harm to the embryo shared by both medical professionals
and patients.[18] Recently, the use of spent culture media (SCM) as an alternative source of embryonic
DNA has been proposed. Research has documented the presence of cf-DNA in SCM, highlighting
its potential for niPGT to evaluate the genetic characteristics of IVF-derived preimplantation
human embryos.[20] Despite this, a standardized noninvasive protocol remains elusive due to the availability
of low sample volume at the start.[21]
[22]
[23] The widespread use of group embryo culture media in IVF clinics further complicates
this, as single embryo culture is crucial for getting spent media specific to each
embryo.[24]
Numerous studies have yielded conflicting findings on the efficacy of group versus
single embryo culture. To address these discrepancies and establish a standardized
noninvasive protocol, the current study used bovine oocytes, which have similar germline-specific
molecular profiles to humans, providing a relevant comparative framework.[20] According to Brouillet et al,[20] SCM can be collected at various preimplantation developmental stages, including
cleaved embryos with fewer than six cells on day 3 and early blastocysts. This provides
further benefits to invasive PGT (iPGT), which relies heavily on embryo biopsy or
blastocentesis. SCM-based PGT offers a viable option for assessing cultured embryos
with diminished implantation potential,[26] which are not suitable for iPGT. SCM-PGT features a rapid turnaround time (less
than 12 h from SCM collection to genetic analysis), potentially providing results
before embryo transfer or cryopreservation.[19]
[27]
The amount of cf-DNA released by embryos varies significantly, impacting ART. Currently,
embryo viability assessment often relies on invasive procedures like embryo biopsy
or PGT, which can be costly, time-consuming, and risky. This underscores the need
for further research to fully understand cf-DNA release by embryos and to optimize
its use in noninvasive embryo assessment.[28]
[29]
Conclusion
In conclusion, the findings of this research highlight the promising potential of
single-drop embryo culture in enhancing the efficiency of noninvasive preimplantation
testing. The observed superiority in blastocyst quality and higher mean conversion
rate suggest that single-drop culture may offer a more effective approach for selecting
embryos with optimal genetic integrity, particularly in cases where genetic disorders
or mutations are of concern. Additionally, the variability in cf-DNA release underscores
the importance of further research to fully understand its implications and optimize
its use in noninvasive embryo assessment. Overall, these findings contribute to the
growing body of evidence supporting the adoption of single-drop culture as a preferred
method for noninvasive preimplantation testing in IVEP.
