TIME LAPSE: ICSI NORMOSPERMIA vs ICSI SEVERO FATTORE MASCHILE C. Lagalla Napoli, 5 7 Ottobre 2017
PRIMO VISION/ PRIMO VISION EVO+ EMBRYOSCOPE/ EMBRYOSCOPE D EEVA GERI MIRI TL EMBRYOSCOPE + 2016
(TEMPI ED INTERVALLI DI TEMPO TRA UNO STADIO CELLULARE E L ALTRO) (MODALITA DI DIVISIONE)
Campbell, Alison. Atlas of Time Lapse Embryology. CRC Press, 2015
PATTERN: DIVISIONI IRREGOLARI 1-3 CLEAVAGES: 2-5 CLEAVAGES: 2-5 2-(3)-5 2-(4)-5 2-(4)-6 PROLONGED S2: REVERSE CLEAVAGE: >4 HRS
Fertilization method (ICSI v IVF) determines kinetics if insemination time is the starting point. Cruz et al., 2013. Dose of rfsh and [E2] on day of hcg affect embryo kinetics. Munoz et al., 2012. Culture technology (medium, ph, O2, etc.)? Ciray et al., 2012 medium, yes Basile et al., 2013 medium, no
: DALLA LETTERATURA ICSI 2,5 h più veloci, ma 4 cell=. FIVET: Higher syncrony FIVET più lente, massimo al 2 cell, ma scompare al 4 cell FIVET più lente fino a blastocisti, Ma la diff. scompare se t0=tfpn
: DALLA LETTERATURA t0= TEMPO DI INSEMINAZIONE: EMBRIONI ICSI PIU VELOCI CRUZ et al., 2013 t0= PNF: NON SI EVIDENZIANO PIÙ DIFFERENZE
: DALLA LETTERATURA BODRI et al., 2015
t0 = t2 : DALLA LETTERATURA
Nagy et al., 1998 OVOCITI INSEMINATI CON: ICSI di spermatozoi testicolari, ICSI di Eiaculato dopo FIVET Non sono state evidenziate differenze fra i primi due gruppi ma è emerso che gli ovociti fecondati con FIVET hanno un ritardo di 4 ore rispetto a quelli fecondati con ICSI
Analizzare l effetto della qualità del seme sulla morfocinetica Analizzare la differenza tra FIVET e ICSI I PARTE 323 embrioni fecondati con ICSI II PARTE 258 embrioni fecondati con IVF + 323 con ICSI
92 coppie 145 PAZIENTI di pazienti PGS (36,0 + 95% CI 35.2 36.8) 581 EMBRIONI ANALIZZATI MORFOCINETICAMENTE CRITERI DI ESCLUSIONE: Pz con < di 3 ovociti inseminati PGD/PGS Inseminazioni con prelievi testicolari VARIABILI MORFOCINETICHE: t0= t ins. t0=pnf (t2, t3, t4, t5, t6, t7, t8, t9) (tm) (tb) (teb) Intervalli: t3 t2; t4 t3, t4 t2 e t8 t4.
Embrioni da ICSI con seme di scarsa qualità (n174) (<3 mil spermatozoi mobili tot e <4% morfologia normale pre trattamento) Embrioni da ICSI con seme di buona qualità (n72) ( >15 mil. per ml MOTILITA TOT > 40% FN >4 pre trattamento)
time (h) ICSI poor semen (n=174) ICSI good semen (n=72) P value tpnf 23.5 (23.0 24.0) 23.0 (22.3 23.7) NS t2 26.5 (25.7 27.3) 25.5 (24.6 26.3) NS t3 37.2 (36.2 38.2) 36.6 (35.6 37.7) NS t4 39.7 (38.3 41.1) 38.6 (37.2 39.9) NS t5 50.4 (48.9 51.9) 49.3 (47.6 50.9) NS t6 53.4 (51.9 55.0) 51.1 (49.3 52.9) NS t7 55.2 (53.7 56.8) 55.4 (52.9 57.8) NS t8 59.3 (57.4 61.2) 58.8 (55.9 61.7) NS t9 72.4 (70.7 74.2) 69.9 (67.4 72.3) NS NESSUNA DIFFERENZA SIGNIFICATIVA tm 90.6 (89.0 92.3) 88.4 (85.9 90.8) NS tb 107.3 (105.4 109.1) 107.9 (104.9 110.9) NS teb 113.6 (111.7 115.5) 112.3 (109.2 115.5) NS E POSSIBILE CONSIDERARE TUTTI GLI EMBRIONI ICSI COME UN UNICO GRUPPO t3 t2 11.4 (10.9 11.9) 11.6 (11.2 12.0) NS t4 t3 3.0 (2.0 3.9) 2.3 (1.4 3.1) NS t4 t2 13.8 (12.7 15.0) 13.6 (12.6 14.5) NS t8 t4 21.4 (19.7 23.1) 21.5 (19.1 24.0) NS Table 1 Kinetics of ICSI embryos developing to blastocyst stage according to good or poor semen sample quality (t0 = time of insemination). Values are mean in hours (95% CI). Student s t test compares the differences between the groups and a P value 0.05 is considered significant.
Le due tecniche di inseminazione a confronto: 258 emb. IVF 323 emb. ICSI t0 = t.ins t0 = PNF per ETA, livello basale di FSH, durata di somministrazione di gonadotropine, dose totale somministrata, % di fertilizzazione, IR e PR.
Table 2a GQE T0=T inseminazione time (h) ICSI (n=135) Standard IVF (n=114) P-value tpnf 22.2 (21.8-22.7) 24.4 (23.9-24.9) 0.000 t2 24.7 (24.3-25.2) 26.9 (26.3-27.4) 0.000 t3 35.9 (35.3-36.4) 37.8 (37.1-38.5) 0.000 t4 36.7 (36.1-37.3) 38.8 (38.1-39.5) 0.000 t5 48.1 (47.2-49.0) 50.9 (49.9-51.9) 0.000 t6 50.0 (49.0-51.0) 52.4 (51.3-53.4) 0.001 t7 51.8 (50.6-53.0) 54.2 (52.9-55.6) 0.009 t8 55.1 (53.6-56.5) 57.6 (56.1-59.1) 0.017 t9 69.8 (68.5-71.2) 72.9 (71.4-74.3) 0.002 tm 86.0 (84.6-87.3) 88.8 (87.4-90.2) 0.005 tb 103.2 (102.0-104.3) 104.7 (103.1-106.3) NS teb 109.3 (108.0-110.5) 110.9 (109.3-112.4) NS t3-t2 11.1 (10.9-11.3) 11.1 (10.8-11.4) NS t4-t3 1.0 (0.7-1.2) 1.0 (0.7-1.4) NS t4-t2 12.0 (11.7-12.3) 12.0 (11.6-12.3) NS t8-t4 18.4 (17.2-19.6) 18.7 (17.5-19.9) NS
Table 2a GQE T0=T inseminazione time (h) ICSI (n=135) Standard IVF (n=114) P-value tpnf 22.2 (21.8-22.7) 24.4 (23.9-24.9) 0.000 t2 24.7 (24.3-25.2) 26.9 (26.3-27.4) 0.000 t3 35.9 (35.3-36.4) 37.8 (37.1-38.5) 0.000 t4 36.7 (36.1-37.3) 38.8 (38.1-39.5) 0.000 t5 48.1 (47.2-49.0) 50.9 (49.9-51.9) 0.000 t6 50.0 (49.0-51.0) 52.4 (51.3-53.4) 0.001 t7 51.8 (50.6-53.0) 54.2 (52.9-55.6) 0.009 t8 55.1 (53.6-56.5) 57.6 (56.1-59.1) 0.017 t9 69.8 (68.5-71.2) 72.9 (71.4-74.3) 0.002 tm 86.0 (84.6-87.3) 88.8 (87.4-90.2) 0.005 tb 103.2 (102.0-104.3) 104.7 (103.1-106.3) NS GLI EMB. FIVET SONO PIU LENTI FINO ALLO STADIO DI MORULA teb 109.3 (108.0-110.5) 110.9 (109.3-112.4) NS t3-t2 11.1 (10.9-11.3) 11.1 (10.8-11.4) NS t4-t3 1.0 (0.7-1.2) 1.0 (0.7-1.4) NS t4-t2 12.0 (11.7-12.3) 12.0 (11.6-12.3) NS t8-t4 18.4 (17.2-19.6) 18.7 (17.5-19.9) NS
Table 2b DE T0= T ins. time (h) ICSI (n=190) Standard IVF (n=144) P-value tpnf 24.0 (23.5-24.6) 26.6 (25.9-27.2) 0.000 t2 27.4 (26.5-28.3) 30.2 (29.1-31.3) 0.000 t3 37.8 (36.7-38.9) 41.5 (40.0-42.9) 0.000 t4 41.2 (39.6-42.8) 43.6 (42.3-44.9) 0.020 t5 51.1 (49.2-53.0) 55.3 (53.3-57.4) 0.003 t6 54.7 (52.8-56.5) 60.9 (58.5-63.3) 0.000 t7 58.5 (56.7-60.3) 64.9 (62.3-67.5) 0.000 t8 63.1 (60.9-65.3) 70.0 (67.0-72.9) 0.002 t9 72.4 (70.1-74.7) 78.6 (75.9-81.3) 0.001 tm 92.5 (90.5-94.5) 96.6 (94.3-98.8) 0.009 tb 112.8 (110.2-115.4) 113.9 (111.4-116.3) NS teb 120.1 (117.3-122.9) 121.0 (118.3-123.8) NS t3-t2 11.7 (11.1-12.3) 12.3 (11.4-13.2) NS t4-t3 4.2 (3.1-5.3) 3.4 (2.5-4.4) NS t4-t2 15.1 (13.8-16.5) 14.3 (13.4-15.2) NS t8-t4 24.9 (22.7-27.0) 27.6 (24.9-30.3) NS
Table 2b - DE time (h) ICSI (n=190) Standard IVF (n=144) P-value tpnf 24.0 (23.5-24.6) 26.6 (25.9-27.2) 0.000 t2 27.4 (26.5-28.3) 30.2 (29.1-31.3) 0.000 t3 37.8 (36.7-38.9) 41.5 (40.0-42.9) 0.000 t4 41.2 (39.6-42.8) 43.6 (42.3-44.9) 0.020 t5 51.1 (49.2-53.0) 55.3 (53.3-57.4) 0.003 t6 54.7 (52.8-56.5) 60.9 (58.5-63.3) 0.000 t7 58.5 (56.7-60.3) 64.9 (62.3-67.5) 0.000 t8 63.1 (60.9-65.3) 70.0 (67.0-72.9) 0.002 t9 72.4 (70.1-74.7) 78.6 (75.9-81.3) 0.001 tm 92.5 (90.5-94.5) 96.6 (94.3-98.8) 0.009 tb 112.8 (110.2-115.4) 113.9 (111.4-116.3) NS GLI EMB. FIVET SONO PIU LENTI FINO ALLO STADIO DI MORULA IN MODO MOLTO PIU SIGNIFICATIVO teb 120.1 (117.3-122.9) 121.0 (118.3-123.8) NS t3-t2 11.7 (11.1-12.3) 12.3 (11.4-13.2) NS t4-t3 4.2 (3.1-5.3) 3.4 (2.5-4.4) NS t4-t2 15.1 (13.8-16.5) 14.3 (13.4-15.2) NS t8-t4 24.9 (22.7-27.0) 27.6 (24.9-30.3) NS
Table 3a- GQE time (h) ICSI (n=135) Standard IVF (n=114) P-value t2 2.5 (2.4-2.6) 2.5 (2.3-2.6) NS t3 13.6 (13.4-13.8) 13.6 (13.3-13.9) NS t4 14.4 (14.1-14.7) 14.5 (14.1-14.8) NS t5 25.8 (25.2-26.5) 26.5 (25.9-27.1) NS t6 27.7 (27.0-28.5) 28.1 (27.4-28.8) NS LE DIFFERENZE SI ANNULLANO t7 29.7 (28.7-30.8) 30.0 (28.9-31.1) NS t8 32.8 (31.5-34.2) 33.2 (31.9-34.5) NS t9 47.6 (46.5-48.8) 48.6 (47.3-49.9) NS tm 63.7 (62.4-65.1) 64.4 (63.1-65.8) NS tb 80.9 (79.8-82.0) 80.3 (78.8-81.8) NS teb 87.1 (85.9-88.2) 86.5 (85.0-88.0) NS
Table 3b - DE time (h) ICSI (n=190) Standard IVF (n=144) P-value t2 3.5 (2.6-4.5) 3.8 (2.9-4.7) NS t3 14.1 (13.1-15.1) 14.7 (13.6-15.9) NS LE DIFFERENZE RIMANGONO: EMB ICSI PIU VELOCI DAL t6 AL t9 t4 17.4 (15.9-19.0) 17.0 (15.9-18.0) NS t5 27.1 (25.4-28.9) 28.5 (26.7-30.3) NS t6 30.9 (29.1-32.6) 34.0 (31.8-36.2) 0.028 t7 35.0 (33.2-36.7) 38.1 (35.7-40.5) 0.035 t8 39.5 (37.3-41.7) 43.2 (40.5-45.9) 0.039 t9 48.8 (46.5-51.0) 52.4 (49.9-55.0) 0.040 tm 69.0 (67.2-70.9) 70.4 (68.3-72.5) NS tb 89.4 (86.9-91.8) 88.4 (86.1-90.7) NS teb 96.7 (94.0-99.5) 95.6 (93.0-98.2) NS
OSSERVAZIONI IN DAY 3 ALLE 66 h 59.1% embrioni di buona qualità (95% CI 53.7% 64.5%) P=0.02 49.6% embrioni di buona qualità (95% CI 43.5% 55.7%)
OSSERVAZIONI IN DAY 3 ALLE 66 h 59.1% di embrioni di buona qualità (95% CI 53.7% 64.5%) OSSERVAZIONI IN DAY 3 ALLE 68 h NS; p=0.12 52.7% di embrioni di buona qualità (95% CI 47.2% 58.1%)
RATE DI BLASTULAZIONE ALLA 120 h: nessuna differenza statistica tra i due gruppi 65.3% di blastocisti (95% CI 60.1% 70.5%) NS; P=0.8 66.3% di blastocisti (95% CI 60.5% 72.1%)
ICSI FIVET
Questa differenza nei tempi può essere spiegata con il fatto che durante la ICSI lo spermatozoo entra direttamente nell ovocita, saltando la penetrazione delle cellule del cumulo, l interazione con la zona pellucida e la fusione con l ovocita. Diversi studi ci suggeriscono quindi che se ovociti fertilizzati con ICSI e con FIVET devono essere analizzati insieme si rende necessario porre come t0 il PNF
Effetto paterno Gli effetti della qualità del seme sullo sviluppo embrionale in seguito a ICSI sono stati discussi da Loutradi et all nel 2006, con uno studio su 1020 embrioni provenienti da 219 coppie. I pazienti sono stati divisi in 5 categorie, in accordo con i parametri del seme. E stata riscontrata una relazione negativa fra la qualità del seme e lo sviluppo embrionale, infatti i tassi di fertilizzazione, di cleavage, la qualità degli embrioni e lo sviluppo a blastocisti calano se la qualità del seme peggiora.
N. TAROZZIR. SCIAJNO R. SCIAJNO N. TAROZZI M.NADALINI F.PENNETTA ANDREA BORINI Direttore scientifico
SEBBENE IL METODO DI INSEMINAZIONE NON ABBIA NESSUN EFFETTO SUGLI OUTCOMES CLINICI, NEL GRUPPO ICSI LA PERCENTUALE DI FORMAZIONE DI BLASTOCISTI E DI EMBRIONI UTILIZZABILI E SIGNIFIVATIVAMENTE INFERIORE A QUELLA FIVET.
Zsolt P.Nagy1, Cecile Janssenswillen, Ronny Janssens, Anick De Vos, Cathe rine Staessen, Hilde Van de Velde and Andre C.Van Steirteghem2 Centre for Reproductive Medicine, University Hospital, Dutchspeaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B 1090 Brussels, Belgium 1Present address: Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy
The results of this study suggest that the timing of pronuclear formation is no different when a testicular spermatozoon is microinjected into the oocytes from when an ejaculated spermatozoon is injected. Secondly, pronuclear development and first cleavage generally take place 4 h sooner after ICSI than after IVF. On the other hand, a higher proportion of oocytes develop two pronuclei asynchronously after IVF than after ICSI.
Abstract In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (>20 106/ml), motility (>30%), and morphology (>50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (<3 106 forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P < 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P < 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols. 1994 Wiley Liss, Inc.
Consequently, no significant difference in fertilization, cleavage and embryo quality rates were observed, when ejaculated or testicular sperm were used for ICSI, while pregnancy and implantation rates after ICSI with testicular sperm were significantly higher than with ejaculated sperm (28). In conclusion, significantly lower oocyte fertilization as well as embryo cleavage and development rates were observed in our study when severely abnormal ejaculated or surgically retrieved sperm were used for ICSI. This, however, does not seem to affect pregnancy and implantation rates to a significant extent, suggesting the implication of maternally derived factors. Overall, a negative relationship was observed between semen quality and embryo development, even before activation of the embryonic genome, suggesting that sperm can affect embryogenesis from a very early stage.