Embed Size (px)
Citation preview
REPRODUCTIVE MEDICINE
Outcomes of human blastocyst transfer after slow-freezing usingsequential culture: a clinical report
Mario Sousa • Mariana Cunha • Paulo Viana •
Joaquina Silva • Jose Teixeira da Silva •
Cristiano Oliveira • Alberto Barros
Received: 1 September 2011 / Accepted: 5 December 2011 / Published online: 20 December 2011
� Springer-Verlag 2011
Abstract
Purpose To present our experience using slow-freezing
from 2005 to 2008, with subsequent newborn outcomes
after transfer of thawed blastocysts.
Methods There were 148 cycles programmed for frozen
blastocyst transfer, which resulted in 142 embryo transfers.
Blastocysts were cultured in sequential media, and pro-
grammed slow-freezing was performed in an apparatus
using a modified Menezo and Veiga method. Thawing
occurred at room temperature under a stream of 5% CO2,
and embryos were transferred about 2 h after thawing.
Results Seventy percent of the blastocysts survived. The
clinical pregnancy rate was 43%, the implantation rate was
27.7% and the rate of live birth was 38%. Twin gestations
occurred in 19.7% of clinical pregnancies, the newborn twin
rate was 6.5% per clinical pregnancy, the male to female
ratio was 1.04, and abortions occurred in 14.8% of clinical
pregnancies. There was one newborn with a 47, XXY
karyotype and another who developed a benign knee tumour.
Conclusion The present results further support that
extended culture to the blastocyst stage and an efficient
freeze–thaw procedure for blastocysts are associated with
high success rates.
Keywords Blastocyst transfer � Slow controlled freezing �Sequential culture � Pregnancy and newborn outcomes
Introduction
The use of an efficient sequential culture method with
prolonged culture times [1, 2] enables the selection of the
best embryos, which are those with the capability to
develop until day 5 or 6 [3–5]. This increases the treatment
efficiency, as blastocysts are associated with higher preg-
nancy rates and have a higher implantation potential [2, 4,
6–9]. These also depend on following a strict blastocyst
Part of this work has been presented at and published in: Silva J,
Cunha M, Viana P, Teixeira da Silva JM, Oliveira C, Goncalves A,
Barros N, Sousa M, Barros A (2010). Retrospective evaluation of the
outcomes of a sequentially cultured human transfer programme by
slow controlled-rate freezing. 26th Annual Meeting of the European
Society of Human Reproduction and Embryology (ESHRE). Rome,
27–30 June, Italy. (Poster). Published: Human Reproduction, 2010, 25
(Suppl 1) i185–186 (P-179).
M. Sousa (&)
Department of Microscopy, Laboratory of Cell Biology, UMIB,
Institute of Biomedical Sciences Abel Salazar (ICBAS),
University of Porto, Lg. Prof. Abel Salazar, 2, 4099-003 Porto,
Portugal
e-mail: [email protected]
M. Cunha � P. Viana � J. Silva � J. T. da Silva � C. Oliveira �A. Barros
Centre for Reproductive Genetics Alberto Barros, Av. do Bessa,
240, 18 Dto. Frente, 4100-012 Porto, Portugal
e-mail: [email protected]
P. Viana
e-mail: [email protected]
J. Silva
e-mail: [email protected]
J. T. da Silva
e-mail: [email protected]
C. Oliveira
e-mail: [email protected]
A. Barros
e-mail: [email protected]; [email protected]
A. Barros
Department of Genetics, Faculty of Medicine,
University of Porto, Porto, Portugal
123
Arch Gynecol Obstet (2012) 285:1473–1478
DOI 10.1007/s00404-011-2174-5
score to enable the correct selection of embryos to transfer
[10–12]. Implantation depends on proper endometrium
preparation, which aims to result in a perfect synchrony
between embryo development and endometrial differenti-
ation [13]; this is best achieved with transfers at the blas-
tocyst stage [2, 3, 8, 14]. The improved implantation rate
for blastocysts reduces the number of embryos necessary to
be transferred, resulting in a reduction of multiple gesta-
tions and an increase in the number of single-embryo
transfers [3, 5–7, 11].
To increase pregnancy and delivery rates, extended
culture is also dependent on a successful blastocyst-freez-
ing programme. The ability to successfully cryopreserve
surplus high-quality blastocysts in a given IVF cycle
without losing significant embryo viability is essential to
maximise the cumulative benefit of a given treatment cycle
[15]. In blastocysts, the cytoplasmic volume of the cells is
low and the ratio of nucleus to cytoplasm is high; also, the
presence of numerous small cells enables a good recovery
in the event that some cells are destroyed during the pro-
cess of freezing–thawing [7, 8, 14].
In our clinic, we perform the slow freezing method
because of the excellent results we are able to obtain. Our
experience with a day 5 embryo-freezing programme was
evaluated retrospectively between 2005 and 2008, and
newborn outcomes are presented here.
Methods
This work did not involve human or animal experiments.
Patient databases were only used with a statistical purpose.
According to the National Law on Medically Assisted
Procreation (PMA, Law 32/2006) and the National Council
on Medically Assisted Procreation (CNPMA, 2008)
guidelines, clinical and laboratorial information were used
after patients’ informed and written consent.
Blastocyst culture and scoring
Blastocysts were scored (1–5) depending on their degree of
development, according to Gardner criteria [11]. The inner
cell mass and trophectoderm were graded (A–C) for those
blastocysts scored as 3 or higher, depending on their cell
number and cohesiveness. Blastocysts scored 1–5 and A–B
were selected for cryopreservation.
Embryos were cultured in two different sequential
media (Medicult and Vitrolife) that were switched every
2 months. For Medicult (Jylling, Denmark) oocytes were
cultured in IVF-medium until microinjection and pronu-
cleus visualisation. Embryos were then cultured in ISM1/
BlastAssist system medium-1 up to day 3 and then stored
in ISM2/BlastAssist system medium-2 until transfer. For
Vitrolife (Kungsbacka, Sweden; serie G3/G5), oocytes
remained in G-IVF-Plus medium until microinjection and
pronucleus visualisation. Thereafter, embryos were cul-
tured in G-1-Plus media to the end of day 3 and then
transferred to G-2-Plus until day 5. Both media contain
human serum albumin (HSA). Medicult uses HEPES buf-
fer, whereas Vitrolife uses MOPS buffer. Vitrolife media
also contains hyaluronan, the concentrations of which
increase from G-1-Plus to G-2-Plus.
Blastocyst slow-freezing and thawing
The Medicult media and methodology (BlastFreeze/Blast-
Thaw) were used for cryopreservation and thawing in all
cases. The freezing and thawing procedure was adapted from
Menezo and Veiga [14] and Virant-Klun et al. [16],
according to Medicult protocols. This is a two-step protocol.
Blastocysts were first frozen in BlastFreeze media pre-
incubated at 37�C and 6% CO2. Blastocysts were placed in
solution-1 (5% glycerol, 0.7 M) for 10 min and then moved
to solution-2 (9% glycerol, 1.4 M; 0.15–0.2 M sucrose) for
another 10 min, both in a humidified incubator with 6% CO2,
5% O2 and 89% N2. Embryos were then loaded into a PETG
clear rigid embryo straw of 0.15 ml and 91 mm (Cryo-Bio-
System, L0Aile, France), under a heated stereomicroscope
(37�C). Programmed slow freezing was performed in a
Planer-K10 apparatus (MiddleSex, UK). Straws were cooled
from room temperature (RT) to -6�C at a rate of 2�C per
minute, stabilised at this temperature for a 30-s soaking
delay, then seeded manually, cooled to -40�C at 0.3�C per
minute, and finally cooled to -150�C at a rate of 35�C per
minute. Thereafter, straws were transferred to liquid nitrogen
(LN2) and stored at -196�C.
Thawing was performed on the day of embryo transfer
at RT, under a bell-shaped glass with a 5% CO2 stream, in
BlastThaw media pre-incubated at 37�C and 6% CO2.
Straws were warmed at RT for 10–15 s and blastocysts
were released into a Petri dish using a drop of solution-1.
They were then incubated for 10 min in the dark, first in
solution-1 (0.4–0.5 M sucrose) and then in solution-2
(0.15–0.2 M sucrose).
Embryos were cultured in a humidified incubator with
6% CO2, 5% O2 and 89% N2, at 37�C. Blastocysts were
transferred into Medicult IVF-medium for 10–20 min and
then transferred to ISM2/BlastAssist system medium-2 for
a minimum of 1–2 h until embryo transfer. For embryo
transfer, blastocysts were placed in UTM-medium con-
taining rHI, HSA and hyaluronan for a mean time of
30 min (range: 0–1 h). For those cases in which embryo
culture was performed using Vitrolife media, embryos
were placed in G-IVF-Plus, then in G2-Plus, and finally in
1474 Arch Gynecol Obstet (2012) 285:1473–1478
123
EmbryoGlue, which contains HSA and hyaluronan. Only
contracted, partially or completely re-expanded blastocysts
with B 50% of cell degeneration were replaced.
Blastocyst transfer
The time from blastocyst thaw to embryo transfer was
2–4 h. Ultrasound-guided embryo transfer was performed
in the large majority of the cases by the same gynecologist,
using a Sure View Wallace Embryo Replacement Catheter
(Smiths Medical Int, Kent, UK). All embryo transfers were
performed in a programmed cycle. The ovulatory cycle
preceding the embryo transfer cycle was controlled to
inhibit ovulation through hormone replacement.
To prepare the endometrium, one oral tablet of 2 mg
estradiol hemihydrate (Isdin, Novo Nordisk, Bagsvaerd,
Denmark) was given every 12 h from day 2 to day 7
(4 mg/day) of the menstrual cycle, followed by 1 tablet every
8 h until day 8 (6 mg/day). An ultrasound was performed by
day 15 of the cycle to evaluate the thickness and echogenicity
of the endometrium. Embryo transfer was programmed
3 days after an ideal endometrial appearance on ultrasound.
Two endovaginal tablets of 100 mg progesterone (Jaba,
Besins Int, Montrouge, France) were added every 8 h
(600 mg/day), beginning 3 days before embryo transfer.
Estradiol and progesterone were maintained until 12 days
after embryo transfer. Embryo transfer occurred mostly
asynchronously, with the blastocyst for transfer being 1 day
older than the endometrium [7]. Implantation was confirmed
by a rise in serum bhCG on days 11–14 following embryo
transfer. A clinical pregnancy was established by ultraso-
nography at 6–7 weeks of gestation.
Results
In our clinic, transfers occur at day 5 whenever possible.
Blastocyst culture depends on the development, quality and
number of available embryos. Generally, we do not pro-
ceed to extended culture when there are three or fewer
embryos of class AB at day 3 [6]. In the large majority of
the cases, thawing was performed for couples who failed to
achieve a pregnancy with fresh blastocysts. The day 5
embryo transfer programme was evaluated retrospectively
from 2005 to 2008. This interval was chosen to allow
follow-up of the children for a full 2 years.
There were 148 cycles programmed for freeze–thaw
blastocyst transfer, representing all age groups and causes of
infertility. Patient characteristics are summarised in Table 1.
Of these, 142 had an embryo transfer (95.9%). The outcomes
of frozen–thawed blastocyst cycles are summarised in
Table 2, and images of blastocyst morphology are shown in
Figs. 1 and 2. There were 264 embryos transferred, with a
mean number of transferred blastocysts of 1.86 (range: 1–3).
Overall, 75% (264/352) of the thawed and recovered
embryos with B 50% of degeneration were transferred. Of
the 142 cycles, 20 had only one embryo available for transfer,
114 had two embryos, and 8 cycles had three embryos.
Grades of degeneration among the 264 embryos transferred
are presented in Table 2.
Table 1 Patient characteristics
Female age: range (mean) 20–42 (32.9)
Male age: range (mean) 26–48 (34.6)
Time of infertility: range (mean) 1–12 (3.7)
Factors of infertility
Female factor (%) 34/148 (23)
Male factor (%) 76/148 (51.4)
Mixed factors (%) 38/148 (25.7)
Origin of the frozen–thawed blastocysts
ICSI (%) 102/148 (68.9)
IVF (%) 39/148 (26.4)
TESE (%) 5/148 (3.4)
PGD (%) 2/148 (1.4)
Reasons for frozen–thawed blastocysts transfer
Failed fresh transfer (%) 131/148 (88.5)
No pregnancy 109/148 (73.6)
Biochemical pregnancy (%) 14/148 (9.5)
Abortion (%) 6/148 (4.1)
Ectopic pregnancy (%) 2/148 (1.4)
Second child (%) 17/148 (11.5)
Table 2 Outcomes of frozen–thawed blastocysts cycles
No. of straws thawed (mean) (range) 220 (1.49)
(1–4)
No. of blastocysts thawed (mean) (range) 508 (3.43)
(1–10)
Post-thaw blastocysts recovery rate (%) 502/508
(98.8)
No. of blastocysts not recovered 6
Post-thaw blastocysts survival rate (%) 352/502
(70.1)
No. of thaw blastocysts with C 50% degeneration 150/502
(29.9)
No. of embryos transferred (mean) 264 (1.86)
No. of embryos transferred with 0% blastomere
degeneration (%)
189/264 (72)
No. of embryos transferred with 1–10% blastomere
degeneration (%)
45/264 (17)
No. of embryos transferred with [10–30%
blastomere degeneration (%)
26/264 (10)
No. of embryos transferred with [30–50%
blastomere degeneration (%)
4/264 (1)
Arch Gynecol Obstet (2012) 285:1473–1478 1475
123
Of the 142 embryo transfer cycles, there were 75 bio-
chemical pregnancies (52.8%), of which 14 did not progress.
There were 61 clinical pregnancies (43%), with 73 sacs and
65 embryos in sacs, of which 48 (78.7%) were singleton
pregnancies, 12 (19.7%) were twin pregnancies, and 1
(1.6%) was ectopic. There were 9 abortions, yielding an
abortion rate of 14.8% per all clinical pregnancies. Of the 61
clinical pregnancies, there were 49 (34.5%) ongoing preg-
nancies. Of the pregnancies that did not continue, there were
nine abortions, one malformed foetus that was removed, and
two clinical pregnancies for which the follow-up was lost.
The implantation rate was 27.7% (73/264).
Fig. 1 Sequential images of the
same embryo after thaw.
Development of an early
blastocyst (BL1), that has
expanded after thaw and culture
until embryo transfer. a time
20 min, BL1. b t 40 min, BL1.
c t 2 h 20 min, BL2.
d t 4 h 20 min, BL4BB. a, bIVF medium. c, d Blast-Assist
System medium-2. Images
taken in an inverted microscope
with Hoffman Optics on a
heated stage (37�C). 9200
(original)
Fig. 2 Two different blastocysts with different developments after
thaw. a BL1 (early blastocyst: blastocel cavity \ 50% embryo
volume), that after thaw (4 h) has not expanded. b BL3AA (full
blastocyst: the blastocel cavity completely fills the embryo), that after
thaw (4 h) developed to BL4AA (expanded blastocyst: the blastocel
cavity is larger than the original embryo volume, and the zona
pellucida is thinning). Blast-Assist System medium-2. Images taken
in an inverted microscope with Hoffman Optics on a heated stage
(37�C). 9200 (original)
1476 Arch Gynecol Obstet (2012) 285:1473–1478
123
There were 49 deliveries: 6 were eutocic and 42 disto-
cic, and one case had missing information. The length of
gestation was mostly normal; 77.6% (38/49) of deliveries
occurred at or after 37 weeks of gestation, and 22.4%
(11/49) of deliveries were preterm. There were 54 new-
borns, yielding a live birth rate of 38% (54/142), with one
case of sex and weight of the child unknown. The rate of
live births per transferred blastocyst was 20% (54/264), and
11% per thawed blastocyst (54/502). 51% of newborns
were male (27/53), and 49% were female (26/53), which
yields a balanced sex ratio of 1.04. The mean weight of the
newborns is presented in Table 3. There were only five
cases of twins delivered, making the newborn twin rate
6.5% (5/61) per clinical pregnancy and 10.2% (5/49) per
delivery. No monozygotic pregnancy was found.
There was one newborn with a 47, XXY karyotype. It
was a distocic delivery at 35 weeks of gestation. The
newborn weighed 1,960 g and was 44.5 cm in length. The
parents were aged 33 (female) and 32 (male) years and had
normal karyotypes. The couple had experienced 1 year of
male factor infertility, and for this reason underwent
intracytoplasmic sperm injection (ICSI). There was one
dichorionic twin pregnancy derived from transfer of blas-
tocysts BL2 and BL4AA. In this twin pregnancy, a foetus,
malformed because of amniotic adhesions, was removed at
18 weeks. The karyotype was 46, XY.
There was one case in which the child developed a
benign knee tumour. Delivery was distocic at 39 weeks of
gestation, from a singleton pregnancy. The female child is
otherwise healthy and was born from an ICSI cycle with a
weight of 3,185 g and a length of 47.5 cm. The parents
were aged 38 (female) and 35 (male) years, had normal
karyotypes, and had experienced three years of mixed-
factor infertility resulting from male, uterine and other
factors.
Discussion
The aim of the present study was to retrospectively eval-
uate our experience with a day 5 embryo slow-freezing
programme from 2005 to 2008, with 142 embryo transfer
cycles.
The success of a blastocyst transfer programme is not
only measured by pregnancy rates obtained with fresh
embryo transfer but also by the successful pregnancy rates
obtained from freezing excess blastocysts. We perform the
slow-freezing method because of the excellent results we
obtain. Slow freezing has the advantages of maintaining a
controlled rate of freezing, in combination with low con-
centrations of cryoprotectants.
Cryo-tolerance is related to the different stages of
development and to blastocyst quality. Blastocysts with
few cells in the inner cell mass and/or in the trophectoderm
have a lower implantation potential in fresh or in freeze–
thaw transfers. Thus, the present good results may be due
to the strict selection of the embryos before cryopreserva-
tion. The developmental characteristics of blastocysts
influence the freezing–thawing outcome. Early blastocysts
have a higher immediate morphological survival, and
expanded and/or hatching blastocysts have more develop-
ment capacity in vitro [7].
Because the mean number of transferred blastocysts was
1.86, the twin rate was high, at 19.7% per clinical preg-
nancy. However, the term twin rate was only 6.5%. Our
results confirm the low birth weight associated with mul-
tiple gestations, with 6 of the 10 twins born weighing less
than 2500 g. For couples, it is difficult when the transfer of
two fresh blastocysts fails, which is the reason why 88.2%
(114/142) of embryo transfers in this study were transfers
of two blastocysts. The number of males and females born
was identical, suggesting that there is no tendency for
gender selection with freeze–thaw blastocysts.
A recent review on slow freezing of human blastocysts
showed that this technique renders excellent results,
although with variable intervals: studies show survival
ranging from 76 to 95%, pregnancy ranging from 31 to
69% and implantation rates ranging from 23 to 43% [8].
The present results confirm and update previous studies
based on blastocyst slow freezing and thawing and suggest
that this technique should not be replaced in centres with
high rates of blastocyst survival, pregnancy, implantation
and birth after thawing.
Acknowledgments Jorge Beires, MD, Gynecologist (Department of
Gynecology and Obstetrics, Unit of Gynecology and Reproductive
Medicine, S. John Hospital, Porto, Portugal) and Jose Manuel
Teixeira da Silva, MD, Gynecologist (oocyte retrieval); Jose Correia,
MD, Anesthesist (Department of Anethesiology, S. John Hospital,
Porto, Portugal); Luis Ferraz, MD, Urologist (Director, Department of
Urology, Hospital Center of Vila Nova de Gaia, Portugal); Paulo
Viana (IVF Lab.), Claudia Osorio, Ana Goncalves and Nuno Barros,
BSc (Lab. Andrology) from Centre for Reproductive Genetics
Alberto Barros. The authors have no connection to any companies or
products mentioned in this article.
Conflict of interest The authors declare that there is no conflict of
interest that could be perceived as prejudicing the impartiality of the
research reported.
Table 3 Birthweight
Single pregnancy: range (mean) 1.960–4.435 g (3.255 g)
C2,500 g (%) 40/43 (93)
\2,500 g (%) 3/43 (7)
Twin pregnancy: range (mean) 0.840–2.960 g (2.019 g)
C2,500 g (%) 4/10 (40)
\ 2,500 g (%) 6/10 (60)
Arch Gynecol Obstet (2012) 285:1473–1478 1477
123
References
1. Gardner DK, Lane M (2002) Development of viable mammalian
embryos in vitro: evolution of sequential media. In: Cibelli JB,
Lanza RP, Campbell KHS (eds) Principles of cloning, chap 9, 1st
edn. Elsevier, USA, pp 187–213
2. Del Marek MA, Langley M, Gardner DK, Confer N, Doody KM,
Doody KJ (1999) Introduction of blastocysts culture and transfer
for all patients in an in vitro fertilization program. Fertil Steril
72:1035–1040. doi:10.1016/50015-0282(99)00409-4
3. Menezo YJR, Veiga A, Torello MJ, Kaufmann R, Hazout A,
Servy EJ (1998) Our 7-year experience in IVF programs with the
human blastocyst: transfer, freezing and biopsy. Fertil Steril
70:S83–S84
4. Gardner DK, Lane M (2000) Embryo culture systems. In: Trou-
son AL, Gardner DK (eds) Handbook of in vitro fertilization,
chap 11, 2nd edn. CRC Press, Boca Raton, pp 205–264
5. Behr B, Gebhardt J, Lyon J, Milki AA (2002) Factors relating to a
successful cryopreserved blastocysts transfer program. Fertil
Steril 77:697–699. doi:10.1016/S0015-0282(01)03267-8
6. Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B (2000)
Comparison of blastocyst transfer with day 3 embryo transfer in
similar patient populations. Fertil Steril 73:126–129. doi:
10.1016/S0015-0282(99)00485-9
7. Van den Abbeel E, Camus M, Verheyen G, Van Waesberghe L,
Devroey P, Van Steirteghem A (2005) Slow controlled-rate
freezing of sequentially cultured human blastocysts: an evalua-
tion of two freezing strategies. Hum Reprod 20:2939–2945. doi:
10.1093/humrep/dei134
8. Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S (2008)
Current aspects of blastocyst cryopreservation. Reprod BioMed
Online 16:311–320 PMID:18284893
9. Surrey E, Keller J, Stevens J, Gustofson R, Minjarez D,
Schoolcraft W (2010) Freeze-all: enhanced outcomes with
cryopreservation at the blastocyst stage versus pronuclear stage
using slow-freeze techniques. Reprod BioMed Online
21:411–417. doi:10.1016/j.rbmo.2010.04.008
10. Menezo YJR, Kaufmann R, Veiga A, Servy EJ (1999) A mini-
atlas of the human blastocyst in vitro. Zygote 7:61–65
PMID:10216918
11. Gardner DK, Lane M, Stevens J, Schlendker T, Schoolcraft WB
(2000) Blastocyst score affects implantation and pregnancy out-
come: towards a single blastocysts transfer. Fertil Steril
73:1155–1158. doi:10.1016/S0015-0282(00)00518-5
12. Veeck LL, Bodine R, Clarke RN et al (2004) High pregnancy rates
can be achieved after freezing and thawing human blastocysts.
Fertil Steril 82:1418–1427. doi:10.1016/j.fertnstert.2004.03.068
13. Murata Y, Oku H, Morimoto Y et al (2005) Freeze thaw pro-
grammes rescue the implantation of day 6 blastocysts. Reprod
BioMed Online 11:428–433 PMID:16274600
14. Menezo Y, Veiga A (1997) Cryopreservation of blastocysts. In:
Proceedings of the 10th world congress on in vitro fertilization
and assisted reproduction, Vancouver, Canada, 24–28 May,
Monduzzi Editore S.p.A., Bologna, Italy, pp 49–53
15. Gardner DK, Lane M, Stevens J, Schoolcraft (2003) Changing the
start temperature and cooling rate in a slow-freezing protocol
increases human blastocysts viability. Fertil Steril 79:407–410.
doi:10.1016/S0015-0282(02)04576-4
16. Virant-Klun I, Tomazevic T, Bacer-Kermavner L, Mivsek J,
Valentincic-Gruden B, Meden-Vrtovec H (2003) Successful
freezing and thawing of blastocysts cultured in sequential media
using a modified method. Fertil Steril 79:1428–1433. doi:
10.1016/S0015-0282(03)00395-9
1478 Arch Gynecol Obstet (2012) 285:1473–1478
123
