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Gonadal activity and chromosomal constitution of in vitro generated embryos
Molecular and Cellular Endocrinology 161 (2000) 111 – 116 www.elsevier.com/locate/mce
Gonadal activity and chromosomal constitution of in vitro generated embryos L. Gianaroli *, M.C. Magli, A.P. Ferraretti, D. Fortini, C. Tabanelli, M. Gergolet S.I.S.ME.R., Reproducti6e Medicine Unit, Via Mazzini 12, 40138 Bologna, Italy
Abstract Chromosomal analysis of pre-implantation embryos was carried out in patients with a poor prognosis of full term pregnancy, which underwent induction of multiple follicular growth. In all, 1034 embryos generated from 191 stimulated cycles were screened for nine chromosome aneuploidy by using the multicolour fluorescence in situ hybridisation technique. Thirty-five percent of the diagnosed embryos were chromosomally normal, whereas the remaining presented with numerical abnormalities, which made them not suitable for transfer. The results obtained confirmed that the incidence of abnormalities is mostly dependent on age; however, monosomy and trisomy are more frequent in poor responders. Accordingly, the pregnancy rate per started cycle was significantly higher in women with a normal response to gonadotropic stimulation (33% vs. 8%, P B0.001). These findings indicate that poor responder patients are physiologically exposed not only to reduced chances of implantation, but also to an increased risk of spontaneous abortion and trisomic pregnancies. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gonadal failure; Multicolour FISH; Poor prognosis; Poor responder; Pre-implantation genetic diagnosis
1. Introduction The notable improvements achieved throughout the years in follicular stimulation protocols, micromanipulation techniques and culture strategies have permitted therapy for an increasing array of infertile couples. Astonishing results have been obtained in the treatment of the extremely severe male factor condition, including azoospermia: after the introduction of the intracytoplasmic sperm microinjection (ICSI) procedure, the chances of pregnancy are in the same range as those resulting in normospermic patients (Van Steirteghem et al., 1993; Gianaroli et al., 1999a). As a general result, an increasing number of cases have being admitted to assisted reproductive techniques. Among them, those diagnosed with a poor prognosis of full term pregnancy are more and more frequent. The term ‘poor prognosis’ refers to a heterogeneous group of patients whose common denominator is represented by the low chances of taking home a baby. This includes women of advanced reproductive * Corresponding author. Tel.: + 39-51-307307; fax: + 39-51302922. E-mail address: [email protected] (L. Gianaroli)
age where the low implantation rate and high incidence of abortion are mostly related to non-disjunctional events during oogenesis. If this is true, the selection of embryos developed from chromosomally normal oocytes is expected to overcome the age effect. Similarly, aneuploidy in morphologically regular embryos could be the cause of the otherwise unexplained repeated unsuccessful attempts in younger patients which experienced multiple in-vitro fertilisation (IVF) failures (Gianaroli et al., 1997a). The preimplantation genetic diagnosis (PGD) of aneuploidy permits to verify the chromosomal status of in vitro generated embryos. The procedure entails the removal of one blastomere from Day 3 morphologically normal embryos and its analysis through the fluorescent in situ hybridisation technique (FISH). Although only a limited number of chromosomes are analysed per single cell, valuable information is obtained regarding the most common aneuploidies in the human. As anticipated, the transfer of euploid embryos is associated to an increased implantation rate thus confirming that the chromosomal constitution has a fundamental role in determining embryo viability (Gianaroli et al., 1999b). The technique is especially advantageous when the selection is performed in a large cohort, to identify
0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 9 ) 0 0 2 3 1 - 2
112
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
the embryos with a normal chromosomal status associated to the best morphological appearance (Gianaroli et al., 1997b). As the frequency of aneuploid embryos in poor prognosis patients is greater than 60%, only one third of the embryos transferred in the absence of FISH results could theoretically be capable of normal implantation. The present study was carried out with the aim of verifying whether the female response to controlled ovarian hyperstimulation has an effect on the chromosomal constitution of the generated embryos and consequently on their implantation potential.
2. Materials and methods
2.1. Patients From September 1996 to August 1999, a total of 135 patients classified as having a poor prognosis of pregnancy attended the S.I.S.ME.R. Reproductive Medicine Unit to undergo 191 assisted conception cycles with PGD of aneuploidy. Indications for PGD were: maternal age 36 years or older (140 cycles); and three or more repeated IVF failures (51 cycles). Induction of multiple follicular growth consisted of a long desensitisation protocol with long-acting GnRH analogues and exogenous gonadotropins according to previously described protocols (Ferraretti et al., 1996). Oocyte retrieval was performed by ultrasound guidance at 36 h post-HCG Table 1 Preimplantation genetic diagnosis for aneuploidy: FISH and clinical results No. cycles Age (M9 S.D.) Previous IVF cycles (M 9S.D.) No. embryos generated
191 37.59 4.0 1.9 91.7 1201
No. embryos FISH analyzed FISH diagnosed (%) FISH normal (%)
1034 1017 (98) 357 (35)
FISH abnormal (%) Monosomic Trisomic Haploid Polyploid Mosaic Multinucleated Anucleated
660 (65) 151 135 31 47 197 24 75
No. cycles transferred No. embryos transferred (M 9S.D.)
134 (70) 2.2 90.9
No. clinical pregnancies (%) On term Ongoing Abortions Ectopic Implantation rate %
48 (36) 33 10 4 1 24.6
Table 2 Overall results in relation to the number of oocytes retrieved 4–7
8–14
]15
No. cycles 20 Age (M9 40.2 92.9 S.D.) No. em30 bryos generated
40 37.4 94.0
85 37.5 94.2
46 36.4 9 3.8
159
583
429
No. embryos FISH diagnosed FISH normal (%) FISH abnormal (%) Monosomic/ trisomic
140
486
363
Retrieved oocytes
1–3
28
9 (32)
45 (32)
177 (36)
126 (35)
19 (68)
95 (68)
309 (64)
237 (65)
13 (68)a
43 (45)
138 (45)
No. cycles transferred
6 (30)b,c
No. clinical pregnancies (%) Abortions Ectopic
2 (33)
3 (14)f
1 0
1 0
Implantation rate %
25.0
22 (55)d,e
7.3g
92 (39)a
68 (80)c,d
38 (83)b,e
26 (36)
17 (45)f
1 0 23.3
1 1 33.7g
a,e
PB0.025. PB0.001. d PB0.01. f PB0.05. g PB0.005. b,c
administration. The collected oocytes were incubated in Earle’s medium supplemented with 10% heat inactivated maternal serum in a 5% CO2 humidified gas atmosphere at 37°C. Insemination was carried out with ICSI or conventional IVF depending on semen indices.
2.2. Assessment of fertilisation and embryo e6aluation Oocytes were checked for the presence of pronuclei and polar bodies at approximately 16 h after insemination. Regularly fertilised oocytes were cultured and examined at 24 and 42 h later. Number and morphology of blastomeres and percentage of fragmentation were recorded. Embryos selected for transfer were replaced into the uterine cavity. Clinical pregnancies were confirmed by the presence of a gestational sac with fetal heart beat. The implantation rate was calculated as the number of gestational sacs with fetal heart beat divided by the total number of embryos transferred.
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
2.3. Embryo biopsy and fluorescence in situ hybridisation technique Embryo biopsy was carried out at 62 – 64 h post-insemination on regularly cleaving embryos. Each embryo was manipulated individually in Hepes-buffered medium; zona opening was achieved using acidic Tyrode’s solution (20 – 22 mm diameter). The biopsied blastomere was transferred to hypotonic solution and fixed in methanol: acetic acid. For the multicolor FISH analysis nine probes were used in a two-step protocol for the simultaneous detection of the chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 (Munne´ et al., 1998). Since December 1998 the probe for chromosome 14 was removed from the panel and replaced by the probe for chromosome 17 in 25 consecutive cycles; from March 1999 the probe specific for chromosome 1 substituted the one specific for chromosome 17. These modifications were made with the aim of identifying other chromosomes beyond XY, 13, 15, 16, 18, 21, and 22, which could play a role in determining aneuploidy at very early stages, and are not detected in clinical pregnancies. The whole procedure required 8 – 10 h; consequently embryo transfer was scheduled on Day 4 (Gianaroli et al., 1999c).
2.4. Statistical analysis Data were analysed by Student’s t-test and x 2 analysis applying the Yates’ correction, 2×2 contingency tables.
113
3. Results As reported in Table 1, 1201 embryos were generated; 1034 of them presented with regular morphology and development, and were selected for embryo biopsy and FISH analysis. The procedure was informative for 1017 embryos (98%); 357 embryos were diagnosed as chromosomally normal (35%), and 660 presented with an abnormal chromosomal complement (65%). In 57 cycles (30%) no FISH normal embryos were detected and embryo transfer was cancelled; in the remaining 134 cycles, 2.29 0.9 euploid embryos were transferred and generated 48 clinical pregnancies (36%) with an implantation rate of 24.6%. Thirty-three pregnancies already delivered, ten are regularly ongoing, and five ended in abortion (four miscarriages (8%) and one ectopic). Patients were arbitrarily divided in four categories according to the number of oocytes retrieved: 1–3; 4–7; 8–14; ] 15. As depicted in Table 2, the frequency of chromosomally abnormal embryos did not differ among the groups, whereas the incidence of monosomy and trisomy events was significantly higher in the first group (68%). The percentage of transferred cycles decreased proportionally with the number of oocytes retrieved. Pregnancy and implantation rates demonstrated a similar trend. According to these results the clinical outcome was strictly dependent on the response to the controlled ovarian hyperstimulation (Fig. 1). The lowest pregnancy and implantation rates were obtained in the poor
Fig. 1. Clinical results according to the number of retrieved oocytes.
114
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
Fig. 2. Pregnancy results in relation to the number of chromosomally normal embryos detected by FISH and the number of collected oocytes in each patient.
responder patients (18 and 10.2%, respectively) compared to the women where eight or more oocytes were retrieved (41% clinical pregnancies and 27.4% implantation rate). Fig. 2 represents the relationship among the number of oocytes collected from each patients, the number of embryos with a normal chromosomal complement, and the clinical pregnancies generated. The highest incidence of pregnancies occurred in the upper-right side of the graph that corresponds to patients presenting with the largest numbers of oocytes. In addition, the implantation rate increased proportionally with the number of euploid embryos per each cohort, from 13.7% with one
chromosomally normal embryo to 32.5% when five or more euploid embryos were detected (Fig. 3). Finally, the results were analysed in relation to the indication for PGD. More abnormal embryos resulted in the maternal age group compared with the category of repeated failures (68% vs. 57%; PB 0.005). As expected, the frequency of monosomy and trisomy was superior in the maternal age group (46% vs. 33% in the group of repeated cycles; P B 0.01), with the highest incidence in the poor responders’ category (72%; Table 3). Accordingly, the percentage of transferred cycles varied in proportion with the total number of collected oocytes in the maternal age group, whereas remained
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
stable in the other group irrespective of the number of oocytes available. In both groups, the pregnancy rate per transferred cycle increased along with the number of collected oocytes.
4. Discussion The response to gonadotropic stimulation for the recruitment of maturing follicles is physiologically related to age up to the termination of ovarian functionality at the time of menopause. The number of oocytes collected after ovulation induction is an indicator of the quality of response to controlled ovarian hyperstimulation. In general, this number decreases proportionally with the increase in the female age, with a concomitant low incidence of implantation and an augmented risk of spontaneous abortion. However, cases of poor response also occur in younger patients; this condition negatively affects the clinical outcome of a treatment cycle.
115
The implementation of PGD of aneuploidy has demonstrated that the poor prognosis condition associated to advanced maternal age and unexplained repeated IVF failures is strictly related to the generation of chromosomally abnormal embryos. In fact, an average of 65% aneuploidy resulted from the analysis performed on a blastomere removed from Day 3 embryos implying that, in these patients, morphological evaluation alone is not sufficient to estimate the potential of regularly developing embryos. Consequently, in the absence of FISH results, the risk of transferring embryos with a poor implantation ability would be too high (Magli et al., 1998). The results derived from the present study indicate that the incidence of chromosomal abnormalities in the embryos yielded by poor responders is the same compared with normal responders. This finding provides an explanation to the low success rate in these patients, where almost two thirds of the few embryos generated are not compatible with implantation. In the category
Fig. 3. Implantation rate associated to the number of FISH detected normal embryos. Table 3 FISH and clinical results in relation to the patients’ indication for PGD ]36 years (no. retrieved oocytes) 1–3
4–7
8–14
No. cycles
19
28
61
No. embryos FISH diagnosed FISH abnormal (%) Monosomy/trisomy (%)
27 18 (67) 13 (72)e
91 67 (73) 36 (54)
No. transferred cycles (%) No. clinical pregnancies % per retrieval % per transfer
6 (32)b,c 2 10 33
12 (43)d,e 3 11f 25
Implantation rate (%)
25.0
14.3
a,f
PB0.05. PB0.005. c PB0.001. b,d,e
]3 IVF failures (no. retrieved oocytes) ]15
]15
1–3
4–7
8–14
1
12
24
270 183 (67) 77 (42)e
1 1 0
49 28 (57) 7 (25)
130 72 (55) 30 (42)
93 54 (58) 15 (28)
49 (80)c,d 19 31 39
26 (81) 12 38f 46
0 0
10 (83) 0
0
0
19 (79) 7 29 37
12 (86) 5 36 42
24.0
35.9
0
0
21.7
29.4
356 237 (67) 108 (46)
32
14
116
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
of maternal age patients, FISH diagnosis contributes a drastic selection of viable embryos; as a consequence a high transfer cancellation rate occurs, but in the presence of at least one euploid embryo pregnancy rates are in the normal range (Table 3). In this way, hopeless embryo transfers are avoided and good chances of implantation result from the replacement of euploid embryos. Poor responders with repeated IVF failures seem to behave differently. Although only a few cases were analysed, FISH selection did not determine any improvement in the clinical outcome. As already observed in previous reports, more than half of the embryos generated from these patients are chromosomally abnormal (Gianaroli et al., 1999b,c). However, the selection of euploid embryos does not increase significantly pregnancy and implantation rates, postulating that other factors strongly contribute to this poor prognosis condition. This is in agreement with the fact that mosaicism and haploidy/polyploidy are the most frequent defects in the screened embryos: disturbances in the mechanisms of cell division, especially centriolar defects and asynchrony between nuclear and cytoplasmic division could play an important role. The analysis of FISH detected abnormalities in the maternal age group revealed that monosomy and trisomy were more frequent in poor responders compared with normal responders. It has been proposed that monosomic embryos either do not implant or are eliminated during the first days of pregnancy (undetected or biochemical pregnancies), on the other hand most of trisomic embryos, including trisomy 21, are destined to spontaneous abortion. Therefore, poor responder patients are not only exposed to low chances of pregnancy but also to an increased risk of abortion and trisomic fetuses. In view of these findings, PGD of aneuploidy assumes a valuable diagnostic importance, although conventional prenatal diagnosis is still recommended in case of a pregnancy. Moreover, this approach permits to increase the chances of implantation by identifying embryos morphologically and chromosomally normal. As expected, PGD is especially beneficial in terms of
percentages of success when the analysis is performed on a large cohort of embryos (Fig. 3). However, the strong reduction in the incidence of spontaneous abortions (Munne´ et al., 1999) and the cancellation of those transfers where no possibility of healthy implantation exists (Gianaroli et al., 1999b) make this procedure a valuable option for giving poor responder patients the best chances of treatment.
References Ferraretti, A.P., Magli, M.C., Feliciani, E., 1996. Relationship of timing agonist administration in the cycle phase to the ovarian response to gonadotropins in the long-down regulation protocols for assisted reproductive technologies. Fertil. Steril. 65, 114–121. Gianaroli, L., Magli, M.C., Munne´, S., et al., 1997a. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod. 12, 1762 – 1767. Gianaroli, L., Magli, M.C., Ferraretti, A.P., et al., 1997b. Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilisation by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril. 68, 1128 – 1131. Gianaroli, L., Magli, M.C., Selman, H., et al., 1999a. Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men. Hum. Reprod. 14, 1034 – 1038. Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al., 1999b. Preimplantation diagnosis for aneuploidies in IVF patients with poor prognosis: identification of the categories to which it should be proposed. Fertil. Steril., 72, 837 – 844. Gianaroli, L., Magli, M.C., Munne´, S., et al., 1999c. Advantages of day four embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J. Assist. Reprod. Genet. 16, 170 – 175. Magli, M.C., Gianaroli, L., Munne´, S., et al., 1998. Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor prognosis patients. J. Assist. Reprod. Genet. 15, 297 – 301. Munne´, S., Magli, M.C., Bahc¸e, M., et al., 1998. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, and 22. Prenatal Diagn. 18, 1459 – 1466. Munne´, S., Magli, M.C., Cohen, J., et al., 1999. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum. Reprod. 14, 2191 – 2199. Van Steirteghem, A., Liu, J., Joris, H., et al., 1993. High fertilisation and implantation rate after ICSI. Hum. Reprod. 8, 1061–1066.
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.
Gonadal activity and chromosomal constitution of in vitro generated embryos L. Gianaroli *, M.C. Magli, A.P. Ferraretti, D. Fortini, C. Tabanelli, M. Gergolet S.I.S.ME.R., Reproducti6e Medicine Unit, Via Mazzini 12, 40138 Bologna, Italy
Abstract Chromosomal analysis of pre-implantation embryos was carried out in patients with a poor prognosis of full term pregnancy, which underwent induction of multiple follicular growth. In all, 1034 embryos generated from 191 stimulated cycles were screened for nine chromosome aneuploidy by using the multicolour fluorescence in situ hybridisation technique. Thirty-five percent of the diagnosed embryos were chromosomally normal, whereas the remaining presented with numerical abnormalities, which made them not suitable for transfer. The results obtained confirmed that the incidence of abnormalities is mostly dependent on age; however, monosomy and trisomy are more frequent in poor responders. Accordingly, the pregnancy rate per started cycle was significantly higher in women with a normal response to gonadotropic stimulation (33% vs. 8%, P B0.001). These findings indicate that poor responder patients are physiologically exposed not only to reduced chances of implantation, but also to an increased risk of spontaneous abortion and trisomic pregnancies. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gonadal failure; Multicolour FISH; Poor prognosis; Poor responder; Pre-implantation genetic diagnosis
1. Introduction The notable improvements achieved throughout the years in follicular stimulation protocols, micromanipulation techniques and culture strategies have permitted therapy for an increasing array of infertile couples. Astonishing results have been obtained in the treatment of the extremely severe male factor condition, including azoospermia: after the introduction of the intracytoplasmic sperm microinjection (ICSI) procedure, the chances of pregnancy are in the same range as those resulting in normospermic patients (Van Steirteghem et al., 1993; Gianaroli et al., 1999a). As a general result, an increasing number of cases have being admitted to assisted reproductive techniques. Among them, those diagnosed with a poor prognosis of full term pregnancy are more and more frequent. The term ‘poor prognosis’ refers to a heterogeneous group of patients whose common denominator is represented by the low chances of taking home a baby. This includes women of advanced reproductive * Corresponding author. Tel.: + 39-51-307307; fax: + 39-51302922. E-mail address: [email protected] (L. Gianaroli)
age where the low implantation rate and high incidence of abortion are mostly related to non-disjunctional events during oogenesis. If this is true, the selection of embryos developed from chromosomally normal oocytes is expected to overcome the age effect. Similarly, aneuploidy in morphologically regular embryos could be the cause of the otherwise unexplained repeated unsuccessful attempts in younger patients which experienced multiple in-vitro fertilisation (IVF) failures (Gianaroli et al., 1997a). The preimplantation genetic diagnosis (PGD) of aneuploidy permits to verify the chromosomal status of in vitro generated embryos. The procedure entails the removal of one blastomere from Day 3 morphologically normal embryos and its analysis through the fluorescent in situ hybridisation technique (FISH). Although only a limited number of chromosomes are analysed per single cell, valuable information is obtained regarding the most common aneuploidies in the human. As anticipated, the transfer of euploid embryos is associated to an increased implantation rate thus confirming that the chromosomal constitution has a fundamental role in determining embryo viability (Gianaroli et al., 1999b). The technique is especially advantageous when the selection is performed in a large cohort, to identify
0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 9 ) 0 0 2 3 1 - 2
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L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
the embryos with a normal chromosomal status associated to the best morphological appearance (Gianaroli et al., 1997b). As the frequency of aneuploid embryos in poor prognosis patients is greater than 60%, only one third of the embryos transferred in the absence of FISH results could theoretically be capable of normal implantation. The present study was carried out with the aim of verifying whether the female response to controlled ovarian hyperstimulation has an effect on the chromosomal constitution of the generated embryos and consequently on their implantation potential.
2. Materials and methods
2.1. Patients From September 1996 to August 1999, a total of 135 patients classified as having a poor prognosis of pregnancy attended the S.I.S.ME.R. Reproductive Medicine Unit to undergo 191 assisted conception cycles with PGD of aneuploidy. Indications for PGD were: maternal age 36 years or older (140 cycles); and three or more repeated IVF failures (51 cycles). Induction of multiple follicular growth consisted of a long desensitisation protocol with long-acting GnRH analogues and exogenous gonadotropins according to previously described protocols (Ferraretti et al., 1996). Oocyte retrieval was performed by ultrasound guidance at 36 h post-HCG Table 1 Preimplantation genetic diagnosis for aneuploidy: FISH and clinical results No. cycles Age (M9 S.D.) Previous IVF cycles (M 9S.D.) No. embryos generated
191 37.59 4.0 1.9 91.7 1201
No. embryos FISH analyzed FISH diagnosed (%) FISH normal (%)
1034 1017 (98) 357 (35)
FISH abnormal (%) Monosomic Trisomic Haploid Polyploid Mosaic Multinucleated Anucleated
660 (65) 151 135 31 47 197 24 75
No. cycles transferred No. embryos transferred (M 9S.D.)
134 (70) 2.2 90.9
No. clinical pregnancies (%) On term Ongoing Abortions Ectopic Implantation rate %
48 (36) 33 10 4 1 24.6
Table 2 Overall results in relation to the number of oocytes retrieved 4–7
8–14
]15
No. cycles 20 Age (M9 40.2 92.9 S.D.) No. em30 bryos generated
40 37.4 94.0
85 37.5 94.2
46 36.4 9 3.8
159
583
429
No. embryos FISH diagnosed FISH normal (%) FISH abnormal (%) Monosomic/ trisomic
140
486
363
Retrieved oocytes
1–3
28
9 (32)
45 (32)
177 (36)
126 (35)
19 (68)
95 (68)
309 (64)
237 (65)
13 (68)a
43 (45)
138 (45)
No. cycles transferred
6 (30)b,c
No. clinical pregnancies (%) Abortions Ectopic
2 (33)
3 (14)f
1 0
1 0
Implantation rate %
25.0
22 (55)d,e
7.3g
92 (39)a
68 (80)c,d
38 (83)b,e
26 (36)
17 (45)f
1 0 23.3
1 1 33.7g
a,e
PB0.025. PB0.001. d PB0.01. f PB0.05. g PB0.005. b,c
administration. The collected oocytes were incubated in Earle’s medium supplemented with 10% heat inactivated maternal serum in a 5% CO2 humidified gas atmosphere at 37°C. Insemination was carried out with ICSI or conventional IVF depending on semen indices.
2.2. Assessment of fertilisation and embryo e6aluation Oocytes were checked for the presence of pronuclei and polar bodies at approximately 16 h after insemination. Regularly fertilised oocytes were cultured and examined at 24 and 42 h later. Number and morphology of blastomeres and percentage of fragmentation were recorded. Embryos selected for transfer were replaced into the uterine cavity. Clinical pregnancies were confirmed by the presence of a gestational sac with fetal heart beat. The implantation rate was calculated as the number of gestational sacs with fetal heart beat divided by the total number of embryos transferred.
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
2.3. Embryo biopsy and fluorescence in situ hybridisation technique Embryo biopsy was carried out at 62 – 64 h post-insemination on regularly cleaving embryos. Each embryo was manipulated individually in Hepes-buffered medium; zona opening was achieved using acidic Tyrode’s solution (20 – 22 mm diameter). The biopsied blastomere was transferred to hypotonic solution and fixed in methanol: acetic acid. For the multicolor FISH analysis nine probes were used in a two-step protocol for the simultaneous detection of the chromosomes XY, 13, 14, 15, 16, 18, 21, and 22 (Munne´ et al., 1998). Since December 1998 the probe for chromosome 14 was removed from the panel and replaced by the probe for chromosome 17 in 25 consecutive cycles; from March 1999 the probe specific for chromosome 1 substituted the one specific for chromosome 17. These modifications were made with the aim of identifying other chromosomes beyond XY, 13, 15, 16, 18, 21, and 22, which could play a role in determining aneuploidy at very early stages, and are not detected in clinical pregnancies. The whole procedure required 8 – 10 h; consequently embryo transfer was scheduled on Day 4 (Gianaroli et al., 1999c).
2.4. Statistical analysis Data were analysed by Student’s t-test and x 2 analysis applying the Yates’ correction, 2×2 contingency tables.
113
3. Results As reported in Table 1, 1201 embryos were generated; 1034 of them presented with regular morphology and development, and were selected for embryo biopsy and FISH analysis. The procedure was informative for 1017 embryos (98%); 357 embryos were diagnosed as chromosomally normal (35%), and 660 presented with an abnormal chromosomal complement (65%). In 57 cycles (30%) no FISH normal embryos were detected and embryo transfer was cancelled; in the remaining 134 cycles, 2.29 0.9 euploid embryos were transferred and generated 48 clinical pregnancies (36%) with an implantation rate of 24.6%. Thirty-three pregnancies already delivered, ten are regularly ongoing, and five ended in abortion (four miscarriages (8%) and one ectopic). Patients were arbitrarily divided in four categories according to the number of oocytes retrieved: 1–3; 4–7; 8–14; ] 15. As depicted in Table 2, the frequency of chromosomally abnormal embryos did not differ among the groups, whereas the incidence of monosomy and trisomy events was significantly higher in the first group (68%). The percentage of transferred cycles decreased proportionally with the number of oocytes retrieved. Pregnancy and implantation rates demonstrated a similar trend. According to these results the clinical outcome was strictly dependent on the response to the controlled ovarian hyperstimulation (Fig. 1). The lowest pregnancy and implantation rates were obtained in the poor
Fig. 1. Clinical results according to the number of retrieved oocytes.
114
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
Fig. 2. Pregnancy results in relation to the number of chromosomally normal embryos detected by FISH and the number of collected oocytes in each patient.
responder patients (18 and 10.2%, respectively) compared to the women where eight or more oocytes were retrieved (41% clinical pregnancies and 27.4% implantation rate). Fig. 2 represents the relationship among the number of oocytes collected from each patients, the number of embryos with a normal chromosomal complement, and the clinical pregnancies generated. The highest incidence of pregnancies occurred in the upper-right side of the graph that corresponds to patients presenting with the largest numbers of oocytes. In addition, the implantation rate increased proportionally with the number of euploid embryos per each cohort, from 13.7% with one
chromosomally normal embryo to 32.5% when five or more euploid embryos were detected (Fig. 3). Finally, the results were analysed in relation to the indication for PGD. More abnormal embryos resulted in the maternal age group compared with the category of repeated failures (68% vs. 57%; PB 0.005). As expected, the frequency of monosomy and trisomy was superior in the maternal age group (46% vs. 33% in the group of repeated cycles; P B 0.01), with the highest incidence in the poor responders’ category (72%; Table 3). Accordingly, the percentage of transferred cycles varied in proportion with the total number of collected oocytes in the maternal age group, whereas remained
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
stable in the other group irrespective of the number of oocytes available. In both groups, the pregnancy rate per transferred cycle increased along with the number of collected oocytes.
4. Discussion The response to gonadotropic stimulation for the recruitment of maturing follicles is physiologically related to age up to the termination of ovarian functionality at the time of menopause. The number of oocytes collected after ovulation induction is an indicator of the quality of response to controlled ovarian hyperstimulation. In general, this number decreases proportionally with the increase in the female age, with a concomitant low incidence of implantation and an augmented risk of spontaneous abortion. However, cases of poor response also occur in younger patients; this condition negatively affects the clinical outcome of a treatment cycle.
115
The implementation of PGD of aneuploidy has demonstrated that the poor prognosis condition associated to advanced maternal age and unexplained repeated IVF failures is strictly related to the generation of chromosomally abnormal embryos. In fact, an average of 65% aneuploidy resulted from the analysis performed on a blastomere removed from Day 3 embryos implying that, in these patients, morphological evaluation alone is not sufficient to estimate the potential of regularly developing embryos. Consequently, in the absence of FISH results, the risk of transferring embryos with a poor implantation ability would be too high (Magli et al., 1998). The results derived from the present study indicate that the incidence of chromosomal abnormalities in the embryos yielded by poor responders is the same compared with normal responders. This finding provides an explanation to the low success rate in these patients, where almost two thirds of the few embryos generated are not compatible with implantation. In the category
Fig. 3. Implantation rate associated to the number of FISH detected normal embryos. Table 3 FISH and clinical results in relation to the patients’ indication for PGD ]36 years (no. retrieved oocytes) 1–3
4–7
8–14
No. cycles
19
28
61
No. embryos FISH diagnosed FISH abnormal (%) Monosomy/trisomy (%)
27 18 (67) 13 (72)e
91 67 (73) 36 (54)
No. transferred cycles (%) No. clinical pregnancies % per retrieval % per transfer
6 (32)b,c 2 10 33
12 (43)d,e 3 11f 25
Implantation rate (%)
25.0
14.3
a,f
PB0.05. PB0.005. c PB0.001. b,d,e
]3 IVF failures (no. retrieved oocytes) ]15
]15
1–3
4–7
8–14
1
12
24
270 183 (67) 77 (42)e
1 1 0
49 28 (57) 7 (25)
130 72 (55) 30 (42)
93 54 (58) 15 (28)
49 (80)c,d 19 31 39
26 (81) 12 38f 46
0 0
10 (83) 0
0
0
19 (79) 7 29 37
12 (86) 5 36 42
24.0
35.9
0
0
21.7
29.4
356 237 (67) 108 (46)
32
14
116
L. Gianaroli et al. / Molecular and Cellular Endocrinology 161 (2000) 111–116
of maternal age patients, FISH diagnosis contributes a drastic selection of viable embryos; as a consequence a high transfer cancellation rate occurs, but in the presence of at least one euploid embryo pregnancy rates are in the normal range (Table 3). In this way, hopeless embryo transfers are avoided and good chances of implantation result from the replacement of euploid embryos. Poor responders with repeated IVF failures seem to behave differently. Although only a few cases were analysed, FISH selection did not determine any improvement in the clinical outcome. As already observed in previous reports, more than half of the embryos generated from these patients are chromosomally abnormal (Gianaroli et al., 1999b,c). However, the selection of euploid embryos does not increase significantly pregnancy and implantation rates, postulating that other factors strongly contribute to this poor prognosis condition. This is in agreement with the fact that mosaicism and haploidy/polyploidy are the most frequent defects in the screened embryos: disturbances in the mechanisms of cell division, especially centriolar defects and asynchrony between nuclear and cytoplasmic division could play an important role. The analysis of FISH detected abnormalities in the maternal age group revealed that monosomy and trisomy were more frequent in poor responders compared with normal responders. It has been proposed that monosomic embryos either do not implant or are eliminated during the first days of pregnancy (undetected or biochemical pregnancies), on the other hand most of trisomic embryos, including trisomy 21, are destined to spontaneous abortion. Therefore, poor responder patients are not only exposed to low chances of pregnancy but also to an increased risk of abortion and trisomic fetuses. In view of these findings, PGD of aneuploidy assumes a valuable diagnostic importance, although conventional prenatal diagnosis is still recommended in case of a pregnancy. Moreover, this approach permits to increase the chances of implantation by identifying embryos morphologically and chromosomally normal. As expected, PGD is especially beneficial in terms of
percentages of success when the analysis is performed on a large cohort of embryos (Fig. 3). However, the strong reduction in the incidence of spontaneous abortions (Munne´ et al., 1999) and the cancellation of those transfers where no possibility of healthy implantation exists (Gianaroli et al., 1999b) make this procedure a valuable option for giving poor responder patients the best chances of treatment.
References Ferraretti, A.P., Magli, M.C., Feliciani, E., 1996. Relationship of timing agonist administration in the cycle phase to the ovarian response to gonadotropins in the long-down regulation protocols for assisted reproductive technologies. Fertil. Steril. 65, 114–121. Gianaroli, L., Magli, M.C., Munne´, S., et al., 1997a. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum. Reprod. 12, 1762 – 1767. Gianaroli, L., Magli, M.C., Ferraretti, A.P., et al., 1997b. Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilisation by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril. 68, 1128 – 1131. Gianaroli, L., Magli, M.C., Selman, H., et al., 1999a. Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men. Hum. Reprod. 14, 1034 – 1038. Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al., 1999b. Preimplantation diagnosis for aneuploidies in IVF patients with poor prognosis: identification of the categories to which it should be proposed. Fertil. Steril., 72, 837 – 844. Gianaroli, L., Magli, M.C., Munne´, S., et al., 1999c. Advantages of day four embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J. Assist. Reprod. Genet. 16, 170 – 175. Magli, M.C., Gianaroli, L., Munne´, S., et al., 1998. Incidence of chromosomal abnormalities from a morphologically normal cohort of embryos in poor prognosis patients. J. Assist. Reprod. Genet. 15, 297 – 301. Munne´, S., Magli, M.C., Bahc¸e, M., et al., 1998. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, and 22. Prenatal Diagn. 18, 1459 – 1466. Munne´, S., Magli, M.C., Cohen, J., et al., 1999. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum. Reprod. 14, 2191 – 2199. Van Steirteghem, A., Liu, J., Joris, H., et al., 1993. High fertilisation and implantation rate after ICSI. Hum. Reprod. 8, 1061–1066.
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