CC BY
25
UDC: 618.2
KHALILI MOHAMMAD A.1, FARAMARZI AZITA1, PALMERINI MARIA GRAZIA2, MACCHIARELLI GUIDO2
MORPHOKINETIC ANALYSIS OF PRE-IMPLANTATION EMBRYOS IN ASSISTED REPRODUCTION PROGRAM
'Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2University of L'Aquila, L'Aquila, Italy.
Research and Clinical Center for Infertility association with multiple pregnancies (1).
Khalili Mohammad A. - PhD, e-mail: ivfkhalili@gmail.com; ^ Multiple pregnancies entail an increased
Faramarzi Azita- Ph.D. Candidate; ^ risk for intrapartum complications as
Department of Life, Health and Environmental Sciences ^a consequence of uterine atony and
Palmerini Maria Grazia - PhD, e-mail: mariagrazia.palmerini@univaq.it; ^ malpresentations, resulting in high
Macchiarelli Guido - MD, Professor. ^ incidences of cesarean sections and
|i postpartum hemorrhage. The financial
ABSTRACT. Embryo selection is a vital part in assisted reproductive technology (ART) programs, with morphology-based grading systems having been widely used for decades. Time-lapse (TL) imaging system combined with embryo morphokinetics can offer a non-invasive means for improving embryo selection for uterine transfer. This approach opens a way to select the best viable embryo non-invasively for single embryo transfer (SET). Thus, implantation potential can be enhanced, while reducing the complications associated with multiple pregnancies.
Key words: morphokinetic analysis, assisted reproduction program, assisted
consequences of multiple pregnancies
^ are substantial for both parents and ^ healthcare providers (1-3). ^ Single-embryo transfer
Single-embryo transfer (SET) is now considered as the preferred ^ practice in IVF cycles to reduce the risk
of adverse outcomes associated with reproductive technology, time-lapse imaging system &
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ¡| multiple gestations. However, in order to
Introduction ^ technology to assess the morphokinetic ^ improve the pregnancy outcomes for SET,
Infertility defined as the inability of ^ performance of embryos which resulted ^ embryologists need reliable biomarkers a couple at reproductive age to achieve a ^ in the desired increase in implantation ^ to aid their selection of the embryos with pregnancy after one year of unprotected ^ rate (3). | the highest developmental potential (4).
intercourse. About 50% of infertile ^ Multiple Pregnancies & ^ Embryo Selection
couples experience inability to conceive ^ complications ^ As SET becomes increasingly applied
due to female factor, 35% to a male In spite of ASRM guidelines to reduce in ART practice, the challenge of identifying
factor, and the remaining fall in the group the number of embryos transferred, the embryo with highest developmental with unexplained infertility (1). For years, ^ many centers still transfer 2-3 embryos ^ competence becomes crucial. Therefore,
assisted techniques referred to as ART despite the increase in risk of multiple there have been investigations in have been used to assist reproduction. ^ pregnancies (3). Multiple pregnancies ^ search of additional viability markers
The technology of in vitro embryo are unfavorable due to the poor neonatal to supplement current criteria for
culture has improved significantly in outcome, maternal complications, long- selection, such as, aneuploidy screening, recent years. However, progress has ^ term developmental problems and high ^ O2 respiration, metabolic profiling and been slow regarding the development ^ costs. Multiple pregnancies are associated ^ gene expression analysis (5, 6). Although,
of reliable methods for assessing with increased rates of stillbirths, these methods are promising, grading embryo developmental competency. ^ neonatal deaths, and infant mortality. ^ systems based on embryo morphology Despite intensive investigations, clinical ^ Multiple gestations also have an increased ^ still remain the preferred method for embryologists still depend on daily ^ morbidity rate due to complications ^ assessing embryonic competence (6).
assessments of developmental rates associated with the increased risk of These morphological assessments are based on cell count on days 2/3, or ^ prematurity, low birth weight. Multiples ^ limited to once a day, since repeated
extent of blastulation on day 5, and also have a compromised long-term removal of embryos from the incubator
morphological features such as degree of outcome, including an increased risk of for observation result in undesired fragmentation and symmetry in cleavage ^ long-term medical and developmental ^ temperature and pH fluctuations in the embryos, or quality of the inner cell mass ^ problems, such as learning problems, ^ culture dish. Embryo development is a (ICM) or trophectoderm in blastocysts (2). ^ cerebral palsy, as well as adult health ^ dynamic event and static observations of
Fortunately, technological developments risks. Also, pregnancy-induced maternal their growth can be limiting in their ability for assessing embryo competence have ^ complications, particularly hypertensive ^ to discern differences between embryos been developed with Time-Lapse (TL) ^ disorders, are more common in ^ at similar cell stages (3, 7, 8).
4.48.2015 a.
Батыс Казахстан медицина журналы
The introduction of TL monitoring ^ morphology. Although all systems use ^ In addition, TL cinematography
systems has allowed more detailed ^ an oil overlay, but differ in the way the ^ has advanced the understanding of
observations on embryo developmental ^ embryos are cultured: the Eeva dish ^ fertilization processes and development
kinetics. The precise timing of specific ^ and the microwell group culture dish for ^ of early human embryos. The opportunity
events, such as pronuclear formation, ^ Primo Vision provide group culturing, in ^ to follow the dynamic developmental
syngamy, early cleavage events, cell cycle ^ which 12 micro-wells (Eeva) and 9-16 ^ patterns of embryos using TL monitoring
intervals, synchronicity of cell division and ^ wells (Primo Vision) share a common ^ will give useful data for embryo selection.
initiation of blastulation are indicators of ^ 50-120 |l volume of medium (Figure 1). ^ TL monitoring also allows a phenomenon
an embryo's developmental potential (7, ^ In contrast, the EmbryoScope provides ^ observed on a specific time point to be
8). The ability to continuously monitor an ^ an individual culture set-up, in which the ^ linked to the embryo's developmental
embryo's progression aids in selecting the culture dish has 12 wells, each holding capacity and implantation achievement.
best embryos for uterine transfer. ^ 20-25 |l of culture medium (2, 3). ^ The TL imaging provides an integrated
Time Lapse Imaging ^ The benefits of time-lapse ^ monitoring system consisting of a safe,
TL analysis was first used ^ technology ^ controlled culture environment which
three decades ago for study of the The first benefit is that the embryos allows for retrospectively detection and
developmental progression of bovine are kept in a less disturbed environment testing for critical events during embryo
embryos (9, 10). Recent interest in TL ^ during culture as they are not exposed ^ development (21, 22).
for assessment of clinical embryos was ^ to changes in gas composition, pH or ^ First time-lapse baby in Iran
engendered following numerous studies ^ temperature shifts, or to the movements ^ Many promising methods for
investigating the potential benefit of that accompany daily embryo evaluation selection of human embryos have been
multi-day scoring of embryos for selection ^ under standard conditions. The second ^ invented, but grading systems based
of the most robust embryo(s) for transfer benefit relates to the additional on morphology criteria remained the
(11). Despite conflicting findings, it is ^ developmental kinetic and phenotypic ^ preferred embryo assessment (23). Close
assumed that with static observations, ^ markers that are acquired as compared ^ relationship between morphological
images captured more frequently will with standard evaluation at distinct time- appearance and developmental
provide substantial information regarding points. It is known that human embryos competence of the embryo at given
the association between morphological ^ do not continuously display morphologic ^ time points is also well documented (23,
development and embryo viability, and ^ features that are currently used during ^ 24). Like many other ART laboratories
that a more developmental kinetics will standard, static morphological grading worldwide, the routine grading systems
be acquired (2). such as, fragmentation (2, 12). are in practice for assessment of zygotes
There are three available TL imaging | The timing of key parameters | and early cleavage embryos in Iran (23,
systems currently used in the embryology ^ from TL monitoring for predictions of ^ 24). Despite efforts to optimize embryo
laboratory, namely Primo Vision, ^ blastocyst formation (13-15), aneuploidy ^ selection methods, there is relatively low
EmbryoScope, and Eeva. They all require (16, 17), and implantation (18-20) have IVF success with a clinical pregnancy rate
the use of a digital inverted microscope ^ been investigated. Various models have ^ of~30% per transfer (23, 24). This often
that acquires images of the embryos at ^ been set based on specific developmental ^ results to the transfer of more than one
preset intervals which are integrated ^ milestones and phenotypes. Several ^ embryo at a time, which increases the risk
to create videos. The EmbryoScope is a retrospective studies have shown that of multiple pregnancies associated with
compact, self-contained incubator with a the use of certain morphokinetic markers neonatal and maternal complications.
built-in camera, while the Eeva and Primo ^ improves prediction of blastocyst ^ Efficient method to reduce the risk of
Vision systems comprise a camera that formation and embryo selection. Also, multiple pregnancies is elective SET with
is placed in a traditional incubator (3-6). ^ higher implantation and pregnancy rates ^ aid of TL monitoring (25).
Each of the systems uses a different light have been reported which has propelled The first Iranian live birth using TL
source and differs in the way the embryos many clinical IVF laboratories to acquire embryo scope to select best embryos
are brought into the field of view (no TL systems (12). Despite the increasing for transfer was reported in August 2015
movement of the embryo culture dish ^ number of studies reporting algorithms ^ (25). A case with tubal factor infertility
vs. constant movement of the dish). The and models for selecting the most was admitted to IVF program with
EmbryoScope and Primo Vision systems viable embryos, it is likely that timing of normozoospermia. After ovarian hyper
use bright field technology that allows the ^ development is primarily determined by ^ stimulation, 6 cumulus oocyte complex
assessment of both kinetic parameters ^ embryo health. Although, other variables ^ (COCs) were retrieved and inseminated
and morphology. While, the dark field ^ of culture condition, use of ICSI, type of ^ with 25,000 progressive sperms/ oocyte.
technology used with Eeva allows the ovarian stimulation protocol, as well as Five zygotes were placed individually into
assessment of kinetic parameters, intrinsic patient characteristics, could the micro wells of equilibrated embryo
but provides limited information on ^ play a role (2). ^ scope dish for a digital TL microscope
Медицинский журнал Западного Казахстана
4.48.2015 а.
Figure 1a. Individual microwells of equilibrated embryoscope dish for time-lapse observation.
Figure 1b. Time-lapse microscope, which is portable for easy setting inside a standard incubator
(Primo Vision, Vitrolife Company, Sweden) observation, and incubated at 37°C, 5% CO2. The following early kinetic markers were assessed: time to 2nd polar body (PB) extrusion, pronuclei (PN) appearance, PN fading or syngamy (tPNf), time to 2 cells (c) (t2), 3c (t3), 4c (t4), 5c (t5), 6c (t6), 7c (t7), and 8c (t8). Durations of the second cell cycle (cc2; t3-t2) and the time to complete synchronous divisions s2 (t4-t3) were calculated. Cleavage anomaly was monitored: direct cleavage (single blastomere divided from 1 to 3 cells). Presence of multinucleation, vaculation, and fragmentation were also recorded On day 3, SET took place based on kinetic parameters of the embryos. Clinical pregnancy was confirmed 7 weeks after SET.
Conclusion
TL gives extra information about embryo growth to embryologists; this data may be used for better selection of embryos for SET, in order to prevent multiple gestations. Continuing monitoring with use of TL lets a more exact identity of embryos that follow likely chromosomally normal. Also, significant events could be assessed retrospectively at any time before embryo selection for transfer. The majority of publications have shown optimism regarding the successful application of TL technology in SET program.
References
1. Keel BA, May JV, De Jonge CJ. Handbook of the assisted reproduction laboratory: CRC PressI Llc; 2000.
2. Racowsky C, Kovacs P & Martins WP. A critical appraisal of time-lapse imaging for embryo selection: where are we and where do we need to go? J Assist Reprod Genet. 2105
3. Desai N, Ploskonka S, Goodman LR, Austin C, Goldberg J & Falcone T. Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles. Reproductive Biology and Endocrinology. 2014;12:54
4. Chen AA, Tan L, Suraj V, Pera RR & Shen S. Biomarkers identified with time-lapse imaging: discovery, validation, and practical application. Fertil Steril. 2013; 99:1035-43.
5. Seli E, Robert C, Sirard MA. OMICS in assisted reproduction: possibilities and pitfalls. Mol Hum Reprod 2010; 16:513-530.
6. Kirkegaard K, Agerholm IE & Ingerslev HJ. Time-lapse monitoring as a tool for clinical embryo assessment. 2012; 27: 1277-1285
7. Chamayou S, Patrizio P, Storaci G, Tomaselli V, Alecci C, Ragolia C, Crescenzo C, Guglielmino A: The use of morphokinetic parameters to select all embryos with full capacity to implant. J Assist Reprod Genet 2013, 30:703-710.
8. Conaghan J, Chen AA, Willman SP, Ivani K, Chenette PE, Boostanfar R, Baker VL, Adamson GD, Abusief ME, Gvakharia M, Loewke KE, Shen S: Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology:
results from a prospective multicenter trial. Fertil Steril 2013, 100:412-419.
9. Massip A, Mulnard J. Time-lapse cinematographic analysis of hatching of normal and frozen-thawed cow blastocysts. J Reprod Fertil. 1980;58:475-8.
10. Massip A, Mulnard J, Vanderzwalmen P, Hanzen C, Ectors F. The behaviour of cow blastocyst in vitro: cinematographic and morphometric analysis. J Anat. 1982;134:399-405.
11. Finn A, Scott L, O'Leary T, Davies D, Hill J. Sequential embryo scoring as a predictor of aneuploidy in poor-prognosis patients. Reprod Biomed Online. 2010;21:381-90.
12. Armstrong S, Vail A, Mastenbroek S, Jordan V, Farquhar C. Timelapse in the IVF-lab: how should we assess potential benefit? Hum Reprod. 2015;30:3-8.
13. Kirkegaard K, Kesmodel US, Hindkjaer JJ, Ingerslev HJ. Timelapse parameters as predictors of blastocyst development and pregnancy outcome in embryos from good prognosis patients: a prospective cohort study. Hum Reprod. 2013;28:2643-52.
14. Milewski R, Kuc P, Kuczynska A, Stankiewicz B, Lukaszuk K, Kuczynski W. A predictive model for blastocyst formation based on morphokinetic parameters in time-lapse monitoring of embryo development. J Assist Reprod Genet. 2015;32:571-9.
15. Chavez SL, Loewke KE, Han J, Moussavi F, Colls P, Munne S, et al.
4.48.2015 œ.
Батыс Казахстан медицина журналы
Dynamic blastomere behaviour reflects | pregnancy. Hum Reprod. 2013;2S:627- | Çali§kan E, Öztel Z, Özay A & Özörnek H.
^ y. ..Mil. ^
human embryo ploidy by the four- | 33. | Reproducibility of a time lapse embryo
cell stage. Nat Commun. 2012;3:1251. | 19. VerMilyea MD, Tan L, Anthony | selection model based on morphokinetic
doi:10.1038/ ncomms2249. | JT, Conaghan J, Ivani K, Gvakharia M, | data in a sequential culture media setting.
16. Cetinkaya M, Pirkevi C, Yelke | et al. Computer-automated time-lapse | J Turk Ger Gynecol Assoc 2014; 15: 156-H, Colakoglu YK, Atayurt Z, Kahraman ^ analysis results correlate with embryo ^ 6°.
S. Relative kinetic expressions defining ^ implantation and clinical pregnancy: ^ 23. Khalili M, Moinia F. Role of cleavage synchronicity are better ^ a blinded, multi-centre study. Reprod ^ embryo morphology and cumulative
predictors of blastocyst formation and | BioMed Online. 2014;29:729-36 | embryo score in pregnancy outcome from
quality than absolute time points. J Assist | 20. Kirkegaard K, Campbell A, | in vitro fertilization and intracytoplasmic
Reprod Genet. 2015;32:27-35. | Agerholm I, Bentin-Ley U, Gabrielsen A, | sperm injection cycles. Mid East Fertil Soc
17. Chawla M, Fakih M, Shunnar | Kirk J, et al. Limitations of a time-lapse | J 2002; 7: 231- 236.
A, Bayram A, Hellani A, Perumal V, et | blastocyst prediction model: a large | 24. Khalili MA, Razavi V, Mardanian
al. Morphokinetic analysis of cleavage | multicentre outcome analysis. Reprod | F, Esfandiari N. The predictive value of stage embryos and its relationship to BioMed Online. 2014;29:156-8. pronuclear morphology screening on
aneuploidy in a retrospective time-lapse 21. Cruz M, Gadea B, Garrido N, embryo development and pregnancy
imaging study. J Assist Reprod Genet. | Pedersen KS, Martínez M, Pérez-Cano I, | outcome in ART cycles. Mid East Fertil Soc
2015;32:69-75. | Muñoz M & Meseguer M. Embryo quality, | J 2008; 13: 203-9.
18. Paternot G, Debrock S, De | blastocyst and ongoing pregnancy rates in | 25. Faramarzi A, Khalili MA, Neubourg D, D'Hooghe TM, Spiessens C. | oocyte donation patients whose embryos | Soleimani M. First successful pregnancies Semi-automated morphometric analysis | were monitored by time-lapse imaging. J | following embryo selection using Time-of human embryos can reveal correlations | Assist Reprod Genet .2011; 28:569-573 | lapse technology in Iran: Case report. Iran between total embryo volume and clinical | 22. Yalgnkaya E, Ergin EG, |j Reprod Med 2015; 13: 253-258.
TYMIH РЕЗЮМЕ
KHALILI MOHAMMAD A.1, FARAMARZI AZITA1, PALMERINI KHALILI MOHAMMAD A.1, FARAMARZI AZITA1, PALMERINI
MARIA GRAZIA2, MACCHIARELLI GUIDO2 | MARIA GRAZIA2, MACCHIARELLI GUIDO2
КОЛДАН ¥РЫК,ТАНДЫРУ БАГДАРЛАМАСЫНДА | МОРФОКИНЕТИЧЕСКИЙ АНАЛИЗ
ИМПЛАНТАЦИЯ АЛДЫНДАГЫ ЭМБРИОНДАРДЬЩ | ПРЕДИМПЛАНТАЦИОННЫХ ЭМБРИОНОВ В ПРОГРАММЕ
МОРФОКИНЕТИКАЛЫК ТАЛДАУЫ | ИСКУССТВЕННОГО ОПЛОДОТВОРЕНИЯ
^едеулт бойынша ^лыми-клиникалык, орталь^ы, ^ Научно-клинический центр по бесплодию,
Шахид Садуги атында^ медицина университетi, Йезд, ^ Медицинский университет имени Шахида Садуги, Йезд,
Иран ^ Иран
2Лакуила университетi, Лакуила, Италия ^ 2Университет Лакуила, Лакуила, Италия
Колдан урык,тандыру технологиясынын (К^Т) ^ Программа технологии искусственного
баедарламасы ондаfан жылдар шамасында кенiнен ^ оплодотворения (ТИО) имеет широкое применение в
крлданылып келед^ сурыпталfан эмбриондар ^ течение десятилетий, где отбор эмбрионов производится
морфологиялык, баfалау жYЙесi негiзiнде eндiрiледi жэне ^ на основе морфологической системы оценок и является
манызды белт болып табылады. Баяулатылfан тYсiрiлiм ^ важной частью. Система замедленной съемки в сочетании
жYЙесi эмбрионнын морфокинетикасымен YЙлесiмiнде ^ с морфокинетикой эмбриона может предложить
жатыр куысына кeшiруге сурыпталfан эмбриондарды ^ неинвазивный метод, чтобы улучшить отбор эмбрионов для
жаксарту Yшiн инвазивтi емес эдкт усына алады. Бул ^ переноса в полость матки. Данный подход открывает путь
тэст бiр тиiмдi эмбрионды кeшiру Yшiн eмiр сYPУге ^ к неинвазивному выбору жизнеспособного эмбриона для
к,абтетп эмбрионнын, инвазивт емес эдiстi тандауына жол ^ переноса одного эффективного эмбриона. Таким образом,
ашады. Осылайша, потенциалды имплантация алдьщ^ы ^ потенциальная имплантация может одновременно снизить
жYктiлiктермен байланысты аск,ынуларды бiр мезгiлде ^ осложнения, связанные с предыдущими беременностями. темендете алады. ^ Ключевые слова: морфокинетический анализ,
Нег'зг'! свздер: морфокинетикалын, талдау, долбан ^ программа искусственного оплодотворения, технология
урык,тандыру багдарламасы, к,олдан урык,тандыру ^ искусственного оплодотворения, система замедленной
технологиясы, баяулатып тYсiру жYйесi. ^ съемки.
МеЗииинский журнал ЗапаЗного Казахстана
4.48.2015 œ.
