COMO MIMAR AL EMBRION

Gloria Caldern, PhD President & Co-Founder Embryotools gloria.calderon@embryotools.com www.embryotools.com Info@embryotools.com

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Factores que Afectan el xito de lasTRA

Diagnostico y buena gua teraputica

Estimulacin ovrica

TEAMWORK

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Procedimientos del FIV Transferencia de embriones Tratamiento de la fase ltea

Puncin folicular

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Y porque afecta tanto el Laboratorio de FIV?

Las mejoras en el laboratorio afectan directamente los resultados de los TRA. Los resultados de los centros de FIV se ven afectados Click edit Master style muyto directamente portitle las variaciones ocurridas en los laboratorios.

Rackowsky et al. 11th World Congress on IVF and Human Reproductive Genetics. Sydney, Australia 1999.

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Control de Calidad
1. Organizacin del Laboratorio. 2. Todo el mundo debe seguir los PNT del Lab. 3. Prestar atencin a cada uno de los miles de detalles del Lab. 4. El personal laboratorio debe someterse a exmenes internos Click to editdel Master title style cada 6 meses.

5. Control diario de los resultados.

6. Sistema de cultivo estable para controlar: A. Temperatura B. pH C. Osmolaridad
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Recursos Humanos
2000 ciclos/ao 6 embrilogos 10 tcnicos

1035 ciclos/ao 13 (10) embriologos Click to edit Master title style

800 ciclos/ao 5 embriologos 6 tcnicos

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Control de Calidad
TIEMPO SUFICIENTE PARA CADA CICLO Suficiente personal de laboratorio para asegurar la mayor atencin durante los procedimientos. CONCENTRACION Cada muestra debe ser tratada individualmente. Click to edit Master title style RESPONSABILIDAD Para la correcta aplicacin de los protocolos en laboratorio.

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Influencia de la Calidad del Aire

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Cohen et al., 1997 Hum Reprod 12:1742-1749

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Influencia de la Calidad del Aire

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Sistemas de cultivo mas utilizados

1.- ABIERTO -Tubo Falcon 5ml - Placas de 4 pocillos - Placas Falcon con pocillo central

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2. CERRADO - Placa de petri: - Microgota overlay o underlay

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Que Debemos Conseguir?

- Control de la Temperatura (alrededor de 37C) Click to edit Master title style

- Control del pH (entre 7,2-7,4) - Control de la Osmolaridad (entre 255-295 mOsm/kg)

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Para

EVITAR CAUSAR STRESS A LOS GAMETOS Y EMBRIONES OBVIANDO AS COMPROMETER Click to edit Master title style SU OPTIMO DESARROLLO IN VITRO.

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Como lo Conseguimos?

Click to edit Master title style

CUIDANDO TODOS LOS DETALLES!!!!!!!!

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Laboratory Factors That Can Affect the Results of ART

Oocyte Prep
Air Quality Handling medium Handling dishes Pipettes Warm surfaces/stages Laminar flow hood Microscope light Culture medium Culture dishes Protein Supplement Culture oil Incubator gas Incubator temp Incubator CO2 PP Equilibration Hyaluronidase Stripping/pipettes
20

Total factors = 175

15

Number of Factors

10

n tio er a sf rv an se -5 Tr pre y 3 g a n o r y D hi C re atc tu H ul C ted y s si ps -3 As Bio y 1 D Da PG re tu ul C SI F V IC v. I p e on r C mP p er Pre tion Sp te llec l y oc o va O m C trie er Re Sp te y oc

Procedure

Provided by Don Rieger PhD

Temperatura
1.- CAMPANAS DE FLUJO LAMINAR
Preferentemente de flujo vertical Con superficie calefactada (temp entre 36,5 y 38C) Click to edit Master title style Velocidad media mientras se trabaja Velocidad alta de noche N suficiente para evitar 2 procedimientos a la vez

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Temperatura
2.- SUPERFICIES CALEFACTADAS En Microscopios Termobloques Mesas calefactadas en quirofano

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Temperatura

3.- ACEITE MINERAL Imprescindible para mantener temperatura, pH y osmolaridad constante durante todos los procedimientos del Click Laboratorio. to edit Master title style Acta como una esponja absorbiendo la mayora de VOC y txicos ambientales protegiendo gametos y embriones.

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Temperatura
A.- Aceite mineral lquido
Mantiene temp, pH y omolaridad estable durante menos tiempo fuera del incubador. Tarda menos horas en equilibrarse

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B.- Aceite mineral denso
Mantiene condiciones de nuestro sistema de cultivo estable durante ms tiempo. Tarda mas horas en equilibrarse
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TEMPERATURE: INTERNAL PROBES

MTG OCTAX LOG & GUARD

18

pH 4.- INCUBADORES
Tradicionales CO2/O2

Desarrollados especialmente para Click to de edit Master title style el cultivo gametos y embriones humanos

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pH

4.- INCUBADORES
Deberemos ajustar el % de CO2 de los incubadores para que el pH del medio de cultivo utilizado este entre 7,2-7,4 que es el pH intracelular. Hay que tener en cuenta condiciones ambientales como altura y humedad. A mayor concentracin de CO2 menor pH

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pH. Internal probes CO2

MTG OCTAX LOG &GUARD

21

Ph

MTG OCTAX LOG & GUARD

22

Research data
PROSPECTIVE AND RANDOMIZED STUDY TO COMPARE TWO pH LEVELS. 7.20-7.25 VS 7.35-7.40
J.M. Molina; S. Carmona; A. Mugica; A. Pellicer; M. Riqueros; M. Esbert; M. Bells; A. Ballesteros; .G. Caldern.

OBJECTIVE To evaluate the effect of culturing embryos in either low pH level (7.20-7.25) or high pH level (7.35-7.40) on IVF cycles outcomes.

group A: low pH level vs group B: high pH level.

Regular patients. (n= 311 embryos) Group A 16.95 % 9.36 % 9.36 % 35.67 % Group B 14.29 % 14.29 % 19.29 % 47.86 % p-value Oocyte recipient patients (n= 530 embryos) Group A 14.40 % 6.40 % 14.00 % 34.80 % Group B 18.21 % 12.14 % 10.00 % 40.35 % NS P=0.017 NS NS p-value

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MN only day 2 MN only day 3 MN day 2 + day 3 Total embryos MN
TABLE 1. Multinucleation rates.

NS* NS

P=0.020 P=0.016

No significant differences were observed regarding clinical pregnancy rate: (Group A: 57.69vs Group B: 47.83 in regular patients and (58.06 vs 56.25) in oocyte recipient patients) nor implantation rate (32.05 vs 34.78 in regular patients and 58.06 vs 56.25 in oocyte recipient patients). However, incidence of multinucleation (MN) was significantly higher in Group B.

TABLE 1. Multinucleation rates.

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pH

5.-Niveles de O2
No hay acuerdo en los niveles de O2 necesarios. Se recomienda entre 5-10% A mayor concentracin de O2 mas rpida recuperacin de las constantes del incubador (menor alteracin de temperatura y pH del medio)
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DOES LOW OXYGEN CONCENTRATION AFFECT BLASTOCYST FORMATION P-853 RATE IN PGD (PREIMPLANTATION GENETIC DIAGNOSIS) CYCLES?
Bells M. 1, Molina J. 1, Riqueros M. 1, Pellicer A. 2, Ballesteros A. 1, Caldern G. 1
1IVI

Barcelona. Ronda General Mitre 14, 08017. Barcelona, Spain. 2IVI Valencia. Plaza de la Polica Local 3, 46015. Valencia. Spain *to correspondence: mbelles@ivi.es

Objective
To evaluate the effect of culturing embryos from PGD cycles till day 5 of development in two different oxygen concentration conditions. N embryos Blastocyst formation (%) Early blastocyst (%) Full blastocyst (%) Hatching blastocyst (%) Good quality (%) Low quality (%) A total of 937 embryos from Preimplantation Genetic Screening (PGS) cycles performed between October 2004 and October 2009 in our clinic were involved in the study. Cases formed two groups: Group 1: embryos cultured in atmospheric (~ 20%) oxygen conditions (138 cycles) Group 2: embryos cultured in low (5%) oxygen concentrations (114 cycles) All embryos were grown in Heracell incubators set up at 37C and 5% CO2. Biopsy of one cell from each good quality embryo (at least 6 even cells with less than 20% fragmentation) were performed on day 3 and transfers occurred on day 5. FISH (Fluorescent In Situ Hybridisation) analysis included chromosomes 13, 15, 16, 17,18, 21, 22, X and Y and no-result rescue were performed when needed. General blastocyst stage and both full and hatching blastocyst quality were assessed based on inner cell mass and trophectoderm characteristics. Data were analyzed by Students group t-test and chisquare analysis as appropriate (p<0,05).

Results
GROUP 1 592 53.6 24 19 21.8 73.8 26.2 GROUP 2 511 49.6 14 23 20.9 84.8 15.2 P=NS P=NS P=0.004 P=NS P=NS P=0.007 P=0.01

Design
Retrospective study of 252 PGD cycles.

Materials and methods

Table 1.- Compares Blastocyst quality of groups 1 and 2.

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No significant differences were found in patients age (40.34.0 vs 39.54.3), number of embryos transferred (ET) (1.60.6 vs 1.70.7), clinical pregnancy per ET (42.7% vs 40.0%), implantation (35.2% vs 35.8%) and miscarriage rate (6.5% vs 5.2%) between the two groups.

Conclusions
The blastocyst formation rate is similar in both groups, as well as the percentage of blastocysts reaching late stages on day 5. However, significantly better embryo development was given in low oxygen concentrations. These results are not reflected in the implantation rate probably because the embryo selection for transfer was made according to chromosomal normalcy.

Bibliography
Marius Meintjes, Samuel J. Chantilis, James D. Douglas, alfred J. Rodriguez, Ali R. Guerami, David M. Bookout, brian D. Barnett, and James D. Madden. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Human Reprod., 2009; vol.24, N 2 pp. 300-307. B Kovacic, V Valisavljevic. Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes. RBM online. June 2008. Vol 17. N 2. 229-236.

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OSMOLARIDAD

Pocos laboratorios tienen en cuenta la Osmolaridad del medio en su sistema de cultivo.

Click to edit Master title style Swain et al (2012) han demostrado que
osmolaridades superiores a 310mOsm/kg afectan el desarrollo embrionario in vitro en el ratn.

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Osmolaridad

1.- Alicuotado de medios 2.- Preparacin de placas de cultivo 3.-to Volumen de latitle gota Click edit Master style 4.- Temperatura

5.- Flujo Laminar

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INCUBATOR PROBLEMS WHEN TOO MANY CYCLES

Embryoscope % fertilization % good quality on Day 3 Age average 82.18 68.41 25.08 Minc 84.68 72.01 25.25 trigas 81.89 61.06* 23.42*

% transfers
% B HCG % clinical pregnancy

100 (24/24)
62.50 (15/24) 54.16 (13/24)

95.83 (23/24)
69.56 (16/23) 65.22 (15/24)

95.83 (23/24)
34.78 (8/23)* 30.43 (7/24)*

% implantation rates
% miscarriage

43.5 (20/46)
15.38 (2/13)

55.6 (25/45)
6.67 (1/15)

24.4 (11/45)*
14.29 (1/7) 28

Medios Cultivo
Necesario utilizar medios de cultivo con aminocidos.(contribuyen en la regulacin de pH y
osmolaridad)

- Medio cultivo nico: Click to editde Master title style - Medios secuenciales:
Deja elegir al embrin lo que necesita en cada momento Dale en cada momento lo que necesita

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Sepulveda et al. Lima, Peru (Fertil. Steril. 91, 1765-70, 2009)

Global ECM/Multiblast
N.S. P = 0.026

% of Zygotes or Transfers

80 60

P = 0.002

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20 0
> 6 cells Day 3 Blastocysts Day 5 Clinical Pregnancy Rate

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Carrillo & Yalcinkaya Wake Forest, NC, USA (J. Clin. Embryol. 13 (4), 35-42, 47, 2010)
Global Quinn's Advantage

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50 25 0 Blastocysts Preg. Rate
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% of Embryos or Transfers

75

P < 0.01

N.S.

N.S.

Impl, Rate
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Wirleitner et al. Bregenz, Austria (Reprod Biomed Online 21, 776-782, 2010)
Global G1/G2

% of Zygotes, Embryos, or Patients

N.S.

50 40

P < 0.05 P < 0.05

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20 10 0 Blastocysts Impl Rate Preg Rate

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Implantation rates in young patients (<36 y/o) with ET on Day 3 and Day 5
MediaX
70% * P < 0.05 60% 50% * P < 0.05

COOK

Click40% to edit Master title style

30%

20%
10%

0%
Day 3 Da3 Day 5 Da5

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Implantation rates in Patients (<36 and 37-39 y/o)

Media X 50% * P < 0.05 COOK

45%
40% 35% 30%to edit Master title style Click 25% 20% 44% 38% 33% 35%

15%
10% 5% 0% Age: <36 Menores 36
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Age: 37-39 37-39

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IVI-BARCELONA January-September 20103(Day 3) GLOBAL

REGULAR Nb CYCLES X AGE X OVOCYTES X EMB. FROZEN. CANCELLED T. CLINICAL PREGNANCY IMPLANTATION % MISCARRIAGE % 333 36,7 9,05 1,6 7,6% 46,3% 33,4% 4,9
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RECIPIENTS 403 41,2 13,9 1,98 3,5 3,1% 58,2% 41,7% 8,6
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EMB. 1,97 Click X to editTRANS. Master title style

IVI-BARCELONA Results: Prospective, Randomized Trial May-July 2011

Nb of cycles

X Age X oocytes Nb cells D2 % fragments D2 Nb cells D3 % fragments D3

Global 50 35,48 0,13 9,44 1,65 3,99 0,97 6,96 3,68 7,96 1,65 6,76 3,88

Global TOTAL 42 36,71 0,6 8,76 1,31 3,81 1,09 6,69 3,09 7,32 1,67 6,48 3,88 53,11% (128/241)
40

p
NS (0,07) NS NS NS p 0,02 NS

Good Quality Embryos Click to edit Master 55,16% title (187/339) style
Nb Transfers
48

NS

X Embryos Transferred
X Embryos Frozen Cancellation Rate Implantation Rate Pregnancy Rate Multiple Preg Rate Miscarriage Rate

2 0,16
1,82 0,86 4% (2/50) 34% 50% (24/48) 29,17% (7/24) 4,17% (1/24)
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2,08 0,18
1,07 0,6 5% (2/42) 31% 45% (18/40) 33,33 (6/18) 5,55% (1/18)
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NS
NS NS NS NS NS NS
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IVI-Barcelona: PGS RESULTS

Period: 01/2005-06/2010 Nb=751 X Age=38.7 4.27
Nb Oocytes (X DS) Nb Biopsied E (%) Nb no informatives(%) Nb abnormal (%) E Transferred (X DS) + BHCG 8525 (11.39 6.46) 3532 (88.79) 47 (1.37) 2157 (63.59) 1.63 0.62 53,91

66.71 Click to Transfers edit Master title style

Clinical pregnancy
Implantation Miscarriage Twin pregnancies Triplets

47,49
40.59 14.77 36.29 1.12
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Aporte Proteico

IMPRESCINDIBLE PARA EL CULTIVO DE EMBRIONES HUMANOS IN VITRO.

It has been suggested that HSA may function to control Click to edit Master title style pH, osmotic pressure and membrane stability, as a carrier of growth factors, as a scavenger of heavy metals and other toxins, and as a source of amino acids (see Blake et al., 2002).

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PGD RESULTS IVI BARCELONA LIFEGLOBAL PROTEIN SUPPLEMENT (LGPS)

Period: 06/2010-07/2011 Nb=242 X Age= 39.29 3.7
Nb Oocytes (X DS)
Nb Biopsied E (%) Nb no informatives(%) Nb abnormal (%) E Transferred (X DS) + BHCG Clinical pregnancy Implantation Miscarriage Twin pregnancies

1906 (9.5 5.6)

1358 (91.3) 29 (2.2) 879 (68.6) 1.63 0.8 68.8 64.3 54.2 17.2 33.3

Transfers 64.2 Click to edit Master title style

Triplets

1.9
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Time Lapse Technology: Embryoscope

More Observations Better Selection

Less Disturbance Better Development

Trigas mixing Air circulation

UV-light decontamination

HEPA particle filter >120 L/hr

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VOC carbon filter

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IVI-BARCELONA Results
Single step culture until the blastocyst stage with or without medium renewal on Day-3
Prospective randomised study with donor oocytes in a time-lapse incubator 628 embryos analysed (ICSI was performed on all oocytes)
In vitro development
Total N. of plates Average of donors age Total number of cultured embryos Blastocysts Blastocyst rates 118 26,7 628 424 67,5 Medium renewal 59 26,7 315 215 68,3 No Medium renewal 59 26,7 313 209 66,8 0,756 (not significant) 0,13 (not significant) p value (Chi-square Test)

Good Quality embryos

Good Quality embryo rates Transferred embryos Percentage of embryos transferred Frozen embryos Percentage of frozen embryos Discarded embryos Percentage of discarded embryos

323
76,2 91 21,5 232 54,7 101 23,8

172
80,0 52 24,2 120 55,8 43 20,0

151
72,2 39 18,7 112 53,6 58 27,8 0,120 (not significant) 0,605 (not significant) 0,184 (not significant)

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IVI-BARCELONA Results
Single step culture until the blastocyst stage with or without medium renewal on Day-3
Prospective randomised study with donor oocytes in a time-lapse incubator
628 embryos analysed (ICSI was performed on all oocytes)
In vivo development
SET or DET same dish No Medium

Number of patients Average of embryos transferred Number of transfers Pregnancy rate Embryos transferred Implantation rates Ongoing Pregnancy rates

Total Medium renewal 59 1.6 58 72.4 91 60.4 88.1 1.5 24 79.2 38 68.4 89.5

renewal

Mixed transfers

1.3 20 75.0 25 60.0 86.7

2.0 14 57.1 28 50.0 87.5

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IVI BARCELONA Results

Single step culture until the blastocyst stage with or without medium renewal on Day-3
Prospective randomised study with donor oocytes in a time-lapse incubator 628 embryos analysed (ICSI was performed on all oocytes)
Timings
t2 (h) t3 (h) cc2 (t3-t2) (h) t4 (h) S2(t4-t3) (h) t5 (h) t9+ (h) tM (h) tMcompact (h) tB (h) tEB (h)

tBHi (h)

Slide Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal Renewal No Renewal

N 273 288 246 256 244 256 266 279 243 247 261 270 234 233 227 232 213 206 199 196 125 119 34 31

Mean 26.3 26.1 36.7 36.2 11.0 10.1 39.0 38.4 2.9 2.5 49.2 48.8 70.8 69.4 82.0 81.2 92.6 92.3 101.8 102.8 109.9 110.8 111.9 110.4

SD 5.3 4.7 6.3 5.5 6.0 3.4 6.9 5.6 3.6 4.1 8.3 7.2 11.9 10.6 11.2 11.2 12.5 12.1 11.9 12.2 8.6 8.7 5.0 4.4

p value (t-test)
0.6792

0.5008
0.1128 0.2687 0.8106

0.5733
0.1856 0.4490 0.8758

0.4147
0.4261 0.7226

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Embryo Development until Blastocyst Stage using EmbryoScope

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Conclusiones

1.- Los sistemas de cultivo abiertos han quedado obsoletos debido a su poca o nula capacidad de regular temperatura, pH y Click to edit Master title style osmolaridad.

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CONCLUSIONES

2.- Dentro de la microgota con aceite, parece mantener mejor las condiciones estables de cultivo el mtodo overlay (Swain 2012)

Click toque edit Master title style 3.Hay prestar mucha atencin a la manipulacin, preparacin y manejo de los medios de cultivo.

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Conclusiones

4.- La estabilidad, reproducibilidad de los medios de cultivo comerciales, junto con la alta estabilidad del sistema de cultivo en microgota, hace imprescindible el tener en cuenta todos los detalles en el title momento Click to edit Master style de la preparacin y elaboracin de las placas de cultivo. Si se ha producido alguna alteracin debido a la mala manipulacin, perdurara durante todo el cultivo in vitro, afectando el desarrollo embrionario.

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Grazie mile Thank You Gracias Obrigado

Gloria Caldern, PhD President & Co-Founder Embryotools gloria.calderon@embryotools.com www.embryotools.com Info@embryotools.com
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