Keywords
niPGT-A - spent culture media - PGT-A - trophectoderm biopsy - blastocyst
Palavras-chave
niPGT-A - meio de cultivo condicionado - PGT-A - biópsia de trofectoderma - blastocisto
Introduction
The technology for in vitro fertilization (IVF) has evolved greatly towards the achievement
of higher success rates, and current state-of-the-art laboratories apply extended
culture to blastocyst stage, vitrification, and time-lapse incubators, for instance.[1] One main issue in reproductive medicine that these technological advances cannot
overcome is advanced maternal age (AMA), which reduces implantation rates due to the
higher frequency of aneuploidies of meiotic origin linked to age.[2]
To address this matter, preimplantation genetic testing for aneuploidies (PGT-A),
performed through a trophectoderm (TE) biopsy and next generation sequencing (NGS),
can be applied. It has been shown to increase implantation rates in AMA patients when
a euploid blastocyst is available for transfer, to decrease miscarriage rates, to
be cost-effective, and to shorten treatment time.[3]
[4]
[5] Nevertheless, recent reports[6]
[7] of deliveries from mosaic/aneuploid blastocysts call into question the reliability
of a TE biopsy to estimate the inner-cell-mass (ICM) ploidy. In addition, TE biopsy
is an invasive procedure that requires expensive specific equipment and highly-trained
embryologists. As a consequence, the biopsy can impair implantation rates if not properly
performed.[8]
[9] In this sense, a non-invasive approach for chromosome screening would be preferred.
Non-invasive PGT-A (niPGT-A) approaches aiming to assess cell-free embryonic DNA in
spent culture media are promising. However, several studies[10]
[11]
[12]
[13]
[14] have reported strikingly variable concordance rates, ranging from 30.3% to 85.7%,
between media and TE samples, which undermines the clinical applicability of this
technology. Interestingly, Huang et al.[15] have achieved a concordance of 93.8% when comparing media and whole embryo results.
All of these previous studies have employed additional manipulation on analyzed embryos,
like vitrification or assisted hatching, prior to the collection of spent culture
media, approaches that would not be applied during an IVF cycle with niPGT-A. In this
sense, Rubio et al.[16] have developed a pilot study aiming to assess the concordance between media and
TE samples from blastocysts that were not submitted to those additional manipulations.
As a result, they have observed an 84% concordance for day-6 or -7 blastocysts.
To fully evaluate this protocol, Rubio et al.[17] have expanded this approach to a prospective multicenter study, including our center
as the Brazilian representative. Until this moment, the analysis of TE and media samples
from 1,301 blastocysts (from 8 IVF centers) has provided an overall concordance of
78.2%,[17] and the delivery of the first baby born in Brazil after niPGT-A.
Case Description
A 35-year-old woman was admitted to our center in 2018 for a first medical consultation
accompanied by her 37-year-old husband. The couple did not present history of infertility,
and they were interested in using IVF and PGT-A in order to avoid congenital anomalies
in the offspring. The wife presented a normal antral follicle count, a serum concentration
of anti-Müllerian hormone of 2.65 ng/mL, and a normal body mass index (BMI = 20.2 kg/m2). The husband presented normal sperm concentration, motility and morphology, and
a BMI of 28.4 kg/m2, which is suggestive of overweight. The sperm DNA fragmentation index of 39% indicated
poor sperm quality. The couple decided to undergo IVF and enroll in our prospective
study assessing the concordance between PGT-A from TE biopsy and niPGT-A from cell-free
DNA in spent culture media.
The patient underwent a gonadotropin-releasing hormone (GnRh) antagonist (Orgalutran)
regimen, with application from days 7 to 10 of the stimulation. Recombinant follicle-stimulating
hormone (FSH; Puregon) was applied from days 1 to 5 on a daily dose of 275 IU, and
human menopausal gonadotropin (hMG; Menopur), from days 5 to 10 on a daily dose of
225 IU. When the dominant follicle reached 18 mm, the patient received a single dose
of human chorionic gonadotropin (hCG; Choriomon). All laboratory material, media,
oil and pipettes used during the treatment were exclusively manipulated by embryologists
wearing gloves, caps, and masks to avoid external DNA contamination. Oocyte pick-up
was performed 36 hours after the administration of hCG, and it resulted in the collection
of 21 cumulus-oocyte complexes. On the date of the aspiration, the seminal sample
had a concentration of 60 million sperm cells/mL, and 65% of progressive motility.
After extensive elimination of corona-cumulus cells to prevent maternal contamination,
18 metaphase-II (MII) oocytes were identified and inseminated by intracytoplasmic
sperm injection (ICSI). After 18 hours of the ICSI, a morphological assessment indicated
18 fertilized oocytes presenting 2 pronuclei and the extrusion of the second polar
body.
The embryos were individually cultured from the pronuclear stage until day 4 in 25
µL droplets of Continuous Single Culture Complete medium (CSCM-C) in low oxygen (5%)
conditions (G185 incubator, K-Systems). At day 4, each embryo was washed in 6 droplets
and transferred to a 10-µL droplet of CSCM-C medium using an individual stripper pipette.
A total of 11 (3 day-5 and 8 day-6) expanded blastocysts were biopsied. For this purpose,
laser zona opening was performed at the time of biopsy, and 5 to 10 TE cells from
the expanded blastocyst were harvested as previously described.[18] After the biopsy, the blastocysts were transferred to a new droplet of medium before
vitrification. The spent culture media (culture from days 4 to 6) from 8 day-6 blastocysts
(blastocysts 4 to 11) were collected for niPGT-A. Blastocysts 1, 2 and 3 were biopsied
in day 5, and their media samples were not analyzed due to the previous low concordance
between PGT-A and niPGT-A results observed for day-5 blastocysts.[16] Both conventional PGT-A and niPGT-A were performed through NGS technology.
Overall, 7 embryos yielded informative results for both TE and media samples, and
1 embryo yielded a non-informative result due to amplification failure from the TE
biopsy ([Table 1]). Among the embryos with informative results, 5 presented concordant diagnosis,
and 2, discordant diagnosis (1 false-positive and 1 false-negative). Blastocysts 4,
6 and 9 presented total concordance between TE and media samples. Blastocyst 7 was
diagnosed as aneuploid by both approaches; however, it exhibited a complementary pattern
(-18q in TE and +18 in medium). Additionally, blastocyst 5 presented partial concordance,
since the monosomy of chromosome 21 was detected only through niPGT-A. Blastocyst
8 was diagnosed as aneuploid based on the TE sample, and as euploid based on the medium
sample (false-negative). On the other hand, blastocyst 10 was diagnosed as euploid
based on the TE sample, and as aneuploid based on the medium sample (false-positive).
Table 1
PGT-A and niPGT-A results
|
Blastocyst
|
PGT-A
|
niPGT-A
|
|
|
4
|
46, XY
|
46, XY
|
Total concordance
|
|
5
|
45, -20, XX
|
45, +20, -21, X0
|
Partial concordance
|
|
6
|
44, -11, -14, XY
|
44, -11, -14, XY
|
Total concordance
|
|
7
|
46, -18q, XY
|
47, +18, XY
|
Partial concordance
|
|
8
|
46, -6, +21, XY
|
46, XY
|
False-negative
|
|
9
|
47, +10, XY
|
47, +10, XY
|
Total concordance
|
|
10
|
46, XX
|
51, +1, +4, +10, +21, +22, XX
|
False-positive
|
|
11
|
Noninformative results
|
Abbreviations: niPGT-A, non-invasive preimplantation genetic testing for aneuploidies;
PGT-A, preimplantation genetic testing for aneuploidies.
In May 2019, the couple decided to undergo their first frozen embryo transfer. The
endometrium was prepared with a daily administration of estradiol valerate (Primogyna)
starting on the second day of the menstrual period. At the 12th day of preparation,
the endometrium had a thickness of 7.5 mm, presenting a triple-line pattern. From
this moment on, the patient received a daily dose of intravaginal progesterone (Utrogestan)
for five days before the transfer. Blastocyst 4, diagnosed as 46, XY by both niPGT-A
from spent culture media and conventional PGT-A from TE biopsy ([Fig. 1]), was warmed up and transferred. After 10 days, beta-hCG quantification yielded
a positive result, and pregnancy developed until 40 weeks with the birth of a healthy
3.8 kg male newborn in February 2020.
Fig. 1 Profiles representing the diagnosis of blastocyst 4 by conventional PGT-A and niPGT-A.
Discussion
Along with the advances in molecular techniques, the clinical widespread application
of niPGT-A is close to a reality in IVF treatments. We report here the first baby
born after niPGT-A in Brazil as part of a multicenter prospective study assessing
the concordance between diagnosis provided by TE biopsy and spent culture media.
In this particular case, the couple was not infertile, and the wife was not of advanced
age, so they did not have a clinical indication for either IVF or PGT-A, and they
were counseled accordingly. Despite the recommendations, they decided to undergo such
treatments due to their personal beliefs and experiences regarding the occurrence
of congenital abnormalities. With the development of niPGT-A technology, we must discuss
its clinical indications and the ethical implications of adding a new diagnostic tool
in the absence of a health problem. Differently from conventional PGT-A, niPGT-A has
the advantage of being completely non-invasive, just like the morphology or morphokinetics
evaluations. None of these non-invasive selection methods causes harm to the embryo,
and they have the same aims: to strengthen embryo selection and decrease the number
of embryo transfers to achieve a live birth.
Three biopsied blastocysts from the couple presented total concordance, while two
presented partial concordance between TE and media samples, which is in agreement
with the results of the study by Rubio et al.[17] In their interim analysis,[17] 8 different centers obtained an overall concordance of 78.2% (range: 72.5% to 86.3%),
a sensitivity of 81.7% (range: 76.5% to 91.3%) and a specificity of 77.4% (range:
64.7% to 87.5%). Interestingly, concordance was not affected by the center where the
testing was conducted, or by the incubator model or culture media used, which supports
the reproducibility of the protocol. For the analysis of the blastocysts, the spent
culture media in contact with the embryo from day 4 to days 6/7 were collected. This
protocol was based on previous studies conducted by Igenomix that reported inferior
concordance rates when using media from day 3 to 5 (33.3%)[12] or day 4 to 5 (63.0%).[16]
The need to extend the blastocyst culture until days 6/7 to obtain reasonable concordance
rates between TE and media samples is a drawback of niPGT-A that must be cautiously
analyzed. While it is generally assumed that the implantation potential of untested
day-6/7 blastocysts is inferior to that of day-5 blastocysts,[19]
[20]
[21]
[22]
[23]
[24] the case for the transfer of euploid blastocysts is still under debate.[25]
[26] In addition, further studies should assess if blastocysts formed only on days 6/7
and those formed on day 5 and maintained in culture until days 6/7 present different
potentials of implantation.
One important obstacle for obtaining low false-negative rates from niPGT-A is the
maternal contamination from corona-cumulus cells that were not completely eliminated
before ICSI. During our validation process, we noticed inferior concordant rates for
patients with cells adhered to the zona pellucida even after oocyte denudation (personal
observation). To solve this problem, we increased the concentration of hyaluronidase
solution, established an incubation of 20 minutes between chemical and mechanical
denudations, and removed all corona-cumulus cells during the mechanical denudation.
Additionally, the use of gloves, caps, masks and exclusive laboratory materials/reagents
is crucial to avoid degradation or contamination of media samples.
The transferred blastocyst 4 was diagnosed as 46, XY by both conventional PGT-A and
niPGT-A, and resulted in the delivery of a healthy newborn. In this sense, a previous
study by Rubio et al.[16] suggested higher ongoing implantation rates for blastocysts with a concordant euploid
diagnosis (52.9%) in comparison to embryos diagnosed as euploid by the TE biopsy and
aneuploid by the spent culture media (16.7%). Additionally, Huang et al.[15] reported a concordance between media and whole blastocysts (93.8%) higher than that
of TE and whole blastocysts (82%) for a set of 50 embryos, suggesting that niPGT-A
would be less biased by the issue of embryonic mosaicism. However, in a recent study,
Rubio et al.[17] found concordances of 84.4% and 87.5% between media and ICM and TE and ICM respectively,
for a set of 80 embryos.
Based on the concordance rate achieved in our own experience, niPGT-A is still not
ready to replace conventional PGT-A, especially for patients who are at risk of having
aneuploid pregnancies (such as AMA patients). In this context, the current feasible
application of niPGT-A would be as an embryo-selection method for patients without
indication for conventional PGT-A. The reliability of ∼ 80% of niPGT-A in the diagnosis
of ploidy diagnosis is superior to that provided by morphological[18]
[27] or morphokinetics evaluations.[28] The benefits of niPGT-A as an embryo-selection method should be demonstrated through
randomized controlled trials.
