- Email: [email protected]
Microsurgical fertilization: world survey 1993
12 Microsurgical fertilization: world survey 1993 YONA T A D I R
Micromanipulation of gametes is not a new field. The early report by Tyler and Monroy (1953) described the difficulty of penetrating the egg membrane with a micropipette. Further experimental studies were designed to clarify mechanisms of fertilization by three main techniques and in various models: (a) injecting hamster sperm directly into the cytoplasm of hamster oocytes (Uehara and Yanagimachi, 1976; Lanzendorf et al, 1988), and mouse sperm into cytochalasin-treated mouse oocytes (Market, 1983); (b) subzonal insemination (SUZI) in mouse oocytes (Barg et al, 1986; Laws-King et al, 1987; Mann, 1988); and (c) breaching the zona pellucida by acid digestion using Tyrode's solution (Gordon and Talansky, 1986) by mechanical means (Malter and Cohen, 1989) or with laser beams (Tadir et al, 1989) to provide an opening through which sperm can gain access to the egg (Figure 1). The move of microsurgical fertilization from basic studies to clinical practice was an expected consequence in view of the low fertilization rate following conventional insemination in vitro in patients with severe malefactor infertility. Preliminary reports on human pregnancy following various kinds of microsurgical fertilization procedures (Ng et al, 1988; Malter and Cohen, 1989; Fishel et al, 1990a) stimulated the clinical use of these procedures for patients who have failed to achieve fertilization in repeated IVF attempts, or for those with sperm quality that does not meet minimal criteria for IVF. However, the lack of clear definitions for male-factor versus severe male-factor infertility (see Chapter 4), and the multivariable nature of sperm malfunction leading to microsurgical fertilization, creates objective difficulties in patient selection and interpretation of the outcome. The first world survey on microsurgical fertilization from 21 different programmes was published in 1991 (Cohen, 1991; see Chapter 4). In this review, 69 clinical pregnancies following partial zona dissection (PZD) (53 babies born) and 59 pregnancies following SUZI (11 babies born) were documented. Although special attention has been devoted in this survey to sperm quality, it was difficult to draw predictive conclusions to guide patient selection. Several recent studies using sibling oocytes supported the recommendation that SUZI and PZD should be practised jointly to improve the overall pregnancy rate (Fishel et al 1990b; Cohen et al, 1992; Tadir et al, 1992). A recent report on improved fertilization and pregnancy rates following intracytoplasmatic sperm injection (ICSI) because of poor sperm quality is encouraging and induced a new wave of optimism (Palermo et al, 1993). Bailli~re's Clinical Obstetrics and Gynaecology-197 Vol. 8, No. 1, March 1994 ISBN 0-7020-1844-9
Copyright © 1994, by Bailli~re Tindall All rights of reproduction in any form reserved
198
v. rAI~Ik
41 centres
Steptoe & Edwards
93 P z e
Ilst baby lVFbornl 1st human Pregnancy and delivery 21 centres
;! .
Tyler & Uehara & Monroy Yanagimachi 1955 A
f /
1976 7/1978 1986-89 A A f I1 suz, , n ] ~
/ I
mouse
- I
ICSl
I
/
~
I [
/ hamster / L sperm-egg J
|
I
survey
Fishel|
1993 Tadir's survey
|
|
in mouse I
2. Gordon e t a l . '
B.B.-I"
"~988"~990• 1991 NG • '1' Cohen's
r Penetrating the 7 / 5 - Bargatal / / egg membrane / / " -Mann.. . [with a micropipetteJJ ] 2. ZD and ] "
~,.164
!
5. ICSI in human
/
5. - Lanzendoff et al. - Palermo et al.
13. SUZl in I / I humanl /
3. Laws-King I ~ ~ ' ~
I
| I
;
I
et ~l. 14, ~'Lu In I
I human I 4. Cohen et al,
Figure 1, Development of micromanipulation techniques.
Although this is not a new approach (Lanzendorf et al, 1988) (see Chapter I), it was not introduced into the routine clinical practice for several reasons.
WORLDWIDE QUESTIONNAIRE In an attempt to update knowledge on current trends in microsurgical fertilization and to focus on patient selection for the various techniques, a clinically-oriented questionnaire was designed and circulated among 53 major IVF centres around the world. Of the 53 centres, 46 responded (87%), of which 41 were actively involved in microsurgical fertilization (mean clinical experience 3.0 + 1.6 (range 0.5-7) years). Contributors are acknowledged for their data and input. The results are presented in a cumulative fashion.
)
199
WORLD SURVEY Table 1. Distribution of various microsurgical fertilization techniques practised in 41 centres in Europe, the USA, Australia and Singapore. No. of clinics practising procedure Continent
SUZI
ICSI
Europe (n = 20)
15 (> 50%)
9 (3 > 90%) 3 (30-40%)
USA (n = 14) Australia and Singapore (n = 7)
7 (:>50%) 6 (> 50%)
2 (25-45%) 3
PZD
Other
8 (4>50%) 1
1 (MIFI') 1 (LZD) 1 (MIFT) 1 (MIFT) 1 (LZD)
MIFT, microinsemination fallopian tube transfer; LZD, laser zona drilling. Percentages are out of total MF cycles performed.
The procedures described in this survey can be categorized in two main groups: (1) sperm injection techniques, i.e. SUZI, ICSI and microinjection fallopian transfer (MIFT); and (2) zona breaching techniques, i.e. PZD (mechanical) and laser zona drilling (LZD). Microdrop insemination was suggested as an alternative technique by 13 programmes. The distribution of the various procedures practised per continent is summarized in Table 1. Comments concerning miscarriages were difficult to interpret. Preliminary data suggested that miscarriage rates following microsurgical fertilization are similar to those following conventional IVF, or are somewhat higher. Patient selection
Twenty-eight centres rely mainly on total motile count (TMC) (Fishel et al, 1992; see Chapter 4) in their selection of patients for microsurgical fertilization. Seven programmes pay special attention to the morphology and few Table 2. Sperm quality selected for microsurgical fertilization.
No. of clinics reporting sperm selection Sperm quality score
TMC
1 2 3 4 5 6 7
< < < < < < >
5000 200 000 0.5 million 1 million 5 million I0 million 10 million
Did not specify No. of clinics practising procedure# Comments Centres reporting pregnancies Centres reporting live births
ICSI
SUZI
PZD
MIFT
LZD
MI
1* 3 4 1 3 1 3
1* 8 1 4 5 4 1 13
-2 3 4 2 4 -2
16
37
17
3
2
13
45 1§ 7 3
33 22
3~ 21 19
1 1
1 1
2 2
TMC, total motile count; MI, microdrop insemination. *No lower limit; tstarted recently; 3:stopped; §waiting for institutional review board.
200
Y. TADIR
rely on Kruger's criteria (Kruger et al, 1988), hemizona assay or the hamster test. The lowest TMC selected by each centre for the various procedures was categorized and scored. Table 2 summarizes the sperm-quality score, the number of clinics reporting sperm quality acceptable for each procedure, the number of clinics that practise each technique and the number of centres that reported clinical pregnancies and live births with each technique. There is a clear distinction between sperm quality selected for ICSI, SUZI and PZD. Poor sperm quality is usually selected for ICSI and SUZI. PZD is practised mainly in the presence of better sperm quality, or in patients who have failed previous IVF attempts in the presence of normal or borderline sperm quality. Two centres reported no lower limit for sperm count acceptable for microfertilization, and that a single sperm in the ejaculate may be sufficient for fertilization.
Pregnancies and babies born The questionnaire emphasized that only microsurgically induced embryos should be included in this registry. This is to avoid inaccurate conclusions concerning trends and results caused by 'mixed embryo transfers' (conventional IVF and microsurgical techniques). Accumulated data from the 41 centres revealed that 971 pregnancies were established following various types of microfertilization procedure and 426 babies were born (Table 3). Most pregnancies (833) and babies born (332) resulted from the various sperm injection techniques.
Malformations Six major malformations were reported of the 426 babies born following microsurgical fertilization (Table 4). A total of 151 karyotypes were performed in utero following the ICSI procedure and all were normal.
Table 3. Number of pregnancies and babies born following microsurgical fertilization.
No. of pregnancies
No. of babies born
SUZI ICSI SUZI + ICSI PZD PZD + SUZI MIFT
453 353"t 15 137 1 12
228 91t 11 93 1 2
Total
971
426
'Mixed' embryo transfers (conventional IVF + microsurgical technique) were not counted. *Two centres updated 20 October 1993; tone centre reported 310 ICSI pregnancies and 88 ICS1 babies born.
201
WORLD SURVEY
Table 4. Malformations reported by 41 centres practising microsurgicalfertilization (of 426 babies born). Procedure
Comments
No malformations
Various
170 normal karyotypes* One trisomy 16
ICSI SUZI
One anecephalus One cleft lip and palate (with duplication of urinary collecting system)? One hydroanecenphalus? Twins with cleft palate?
PZD Combined SUZI + ICSI
Reported by 24 centres (who had live births) Performed in utero Miscarried Out of triplet (two normal)
ICSI ICSI
Out of triplet (two normal) Both twins
*Updated by two centres 20 October 1993; ?updated by one centre 7 October 1993.
Although these results should still be considered somewhat preliminary, they are encouraging.
Tips for beginners Fifteen team leaders responded in a similar term 'experience, experience, experience' to the question: 'What message do you have for colleagues who are in the process of setting up an MM facility?' Others stressed the importance of dedicated staff, good equipment and 'start ICSI'. Four centres (two that do not practise microsurgical fertilization) suggested: 'wait for better results'.
Envoi It is too early to predict what the impact of new micromanipulation techniques on microsurgical fertilization may be. The recent trend towards sperm injection casts a shadow on the use of drilling techniques as treatment for severe male-factor infertility. Time will tell what is the grey zone in which microsurgical fertilization and conventional non-manipulated techniques compete or complement each other. As previously stated, several programmes pay special attention to some other selection methods in addition to TMC. It is not yet clear what the predictive value of these tests is, or the criteria for patient selection. However, there is little doubt that the ejaculated specimen with few motile (or even non-motile) sperm may benefit from a micromanipulating or injection technique. The information gathered in this survey is encouraging not only because of the growing numbers of pregnancies and live births that are being established, but also the relatively small number of congenital malformations. This issue is still a source for major concern as more aggressive intervention in the process of conception takes place. Improved fertilization and pregnancy rates following microsurgical fertilization will expand the spectrum of indications for these procedures.
202
Y. TADIR
Acknowledgements This work was supported by grants NIH 2RO1 CA32248 and 5P41 RR01192, DOE DE-FG0391ER61227 and ONR N00014-91-C-0134. The following people contributed to the survey: A. Acosta, S. AI Hasani, S. Amit, R.H. Asch, J.P. Balmaceda, D. Barlow, P.N. Barri, P. Brindsen, M. Bustillo, J. Cheek, J. Cohen, I. Craft, P. Dellenbach, P. Devroi, K. Diedrich, J. Donnez, J. Dor, W. Feichtinger, B. Fisch, S. Fishel, J. Garcia, L. Gianaroli, M. Glezerman, J. Gordon, S. Gordts, L. Hambereger, G. Hartshorne, B. Hedon, J. Hennessey, C. Hi, D.L. Hill, W.J. Holmgren, V. Insler, J. Itzkovitz, I. Johnston, A.M. Junca, P. Katayama, R. Kazer, J. Mandelbaum, S. Mashiach, P.L. Matson, L. Metier, D. Mortimer, S.-C. Ng, A. Obruca, T. Ord, J. Peek, I. Pike, J.P. Ryan, J. Salat Baroux, D. Saunders, R. Schoysman, J.D. Schulman, P. Serafini, J. Stangel, E. Stehlik, A. Trounson, M. Tucker, J. Tyler, P. Vanderzwalmen, A. Van Steirteghem, L.L. Veeck, A. Veiga, Y. Verlinsky, G. Warnes, A. Wittemer, D. Yang, B. Yee, Y.K. Ting.
REFERENCES Barg PE, Wahrman MZ, Talansky BE & Gordon JW (1986) Capacitated, acrosome reacted but immotile sperm, when microinjected under the zona pellucida, will not fertilize the oocyte. Journal of Experimental Zoology 237: 365-374. Cohen J (1991) A review of clinical microsurgical fertilization. In Cohen J, Malter EH, Talansky BE & Grifo J (eds) Micromanipulation of Human Gametes and Embryos, pp 163-190. Cohen J, Alikani M, Adler A et al (1992) Microsurgical fertilization procedures: the absence of stringent criteria for patient selection. Journal of Assisted Reproduction and Genetics 9: 197-206. Fishel S, Antoniori S, Jackson P e t al (1990a) Twin birth after subzonal insemination. Lancet 335: 722-723. Fishel S, Antoniori S, Jackson P e t al (1990b) Subzonal insemination for the alleviation of infertility. Fertility and Sterility 54: 828-835. Fishel S, Antinori S, Jackson P e t al (1991) Presentation of six pregnancies established by subzonal insemination (SUZI) Human Reproduction 6: 124-130. Fishel S, Timson J, Lisi F & Rinaldi L (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertility and Sterility 57: 840-849. Gordon JW & Talansky BE (1986) Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. Journal of Experimental Zoology 239: 347-354. Kruger TF, Acosta AA, Simmons KF et al (1988) Predictive value of abnormal morphology in in vitro fertilization. Fertility and Sterility 49:112-117. Lanzendorf SE, Maloney MK, Veek LL et al (1988) A preclinical evaluation of pronuclear formation by microinjection of spermatozoa into the human oocyte. Fertility and Sterility 49: 835-842. Laws-King A, Trounson A, Sathananthan H & Kola I (1987) Fertilization of the human oocyte by microinjection of single sperm under the zona pellucida. Fertility and Sterility 48: 637642. Malter EH & Cohen J (1989) Partial zona dissection of the human oocyte: a non-traumatic method using micromanipulation to assist zona pellucida penetration. Fertility and Sterility 51: 139-148. Mann JR (1988) Full term development of mouse eggs fertilized by a spermatozoon microinjected under the zona pellucida. Biology of Reproduction 38: 1077-1083. Market CL (1983) Fertilization of mammalian eggs by sperm injection. Journal of Experimental Zoology 228: 195-201. Ng S-C, Bongso A, Ratnam SS et al (1988) Pregnancy after transfer of sperm under zona. Lancet 332:790 (letter). Palermo G, Joris H, Derde MP et al (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmatic sperm injection. Fertility and Sterility 59: 826--835. Tadir Y, Wright WH & Berns MW (1989) Cell micromanipulation with laser beams. In Capi-
WORLD SURVEY
203
tanio GL, Asch RH, De Cecco L & Croce S (eds) G.I.F.T.: From Basics to Clinics pp 359368. New York: Raven Press. Tadir Y, Amit S, Rufas O et al (1992) Assisted fertilization by sub-zonal sperm insertion and partial zona dissection: comparative analysis related to sperm quality. Presented at the 48th Annual Meeting of the American Fertility Society. Fertility and Sterility Program Supplement p.s.-62. Tyler A & Monroy A (1953) Apparent and real micro-injection of echinoderm eggs. Journal of Experimental Biology 109: 370. Uehara T & Yanagimachi R (1976) Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biology of Reproduction 15: 467-470.
Micromanipulation of gametes is not a new field. The early report by Tyler and Monroy (1953) described the difficulty of penetrating the egg membrane with a micropipette. Further experimental studies were designed to clarify mechanisms of fertilization by three main techniques and in various models: (a) injecting hamster sperm directly into the cytoplasm of hamster oocytes (Uehara and Yanagimachi, 1976; Lanzendorf et al, 1988), and mouse sperm into cytochalasin-treated mouse oocytes (Market, 1983); (b) subzonal insemination (SUZI) in mouse oocytes (Barg et al, 1986; Laws-King et al, 1987; Mann, 1988); and (c) breaching the zona pellucida by acid digestion using Tyrode's solution (Gordon and Talansky, 1986) by mechanical means (Malter and Cohen, 1989) or with laser beams (Tadir et al, 1989) to provide an opening through which sperm can gain access to the egg (Figure 1). The move of microsurgical fertilization from basic studies to clinical practice was an expected consequence in view of the low fertilization rate following conventional insemination in vitro in patients with severe malefactor infertility. Preliminary reports on human pregnancy following various kinds of microsurgical fertilization procedures (Ng et al, 1988; Malter and Cohen, 1989; Fishel et al, 1990a) stimulated the clinical use of these procedures for patients who have failed to achieve fertilization in repeated IVF attempts, or for those with sperm quality that does not meet minimal criteria for IVF. However, the lack of clear definitions for male-factor versus severe male-factor infertility (see Chapter 4), and the multivariable nature of sperm malfunction leading to microsurgical fertilization, creates objective difficulties in patient selection and interpretation of the outcome. The first world survey on microsurgical fertilization from 21 different programmes was published in 1991 (Cohen, 1991; see Chapter 4). In this review, 69 clinical pregnancies following partial zona dissection (PZD) (53 babies born) and 59 pregnancies following SUZI (11 babies born) were documented. Although special attention has been devoted in this survey to sperm quality, it was difficult to draw predictive conclusions to guide patient selection. Several recent studies using sibling oocytes supported the recommendation that SUZI and PZD should be practised jointly to improve the overall pregnancy rate (Fishel et al 1990b; Cohen et al, 1992; Tadir et al, 1992). A recent report on improved fertilization and pregnancy rates following intracytoplasmatic sperm injection (ICSI) because of poor sperm quality is encouraging and induced a new wave of optimism (Palermo et al, 1993). Bailli~re's Clinical Obstetrics and Gynaecology-197 Vol. 8, No. 1, March 1994 ISBN 0-7020-1844-9
Copyright © 1994, by Bailli~re Tindall All rights of reproduction in any form reserved
198
v. rAI~Ik
41 centres
Steptoe & Edwards
93 P z e
Ilst baby lVFbornl 1st human Pregnancy and delivery 21 centres
;! .
Tyler & Uehara & Monroy Yanagimachi 1955 A
f /
1976 7/1978 1986-89 A A f I1 suz, , n ] ~
/ I
mouse
- I
ICSl
I
/
~
I [
/ hamster / L sperm-egg J
|
I
survey
Fishel|
1993 Tadir's survey
|
|
in mouse I
2. Gordon e t a l . '
B.B.-I"
"~988"~990• 1991 NG • '1' Cohen's
r Penetrating the 7 / 5 - Bargatal / / egg membrane / / " -Mann.. . [with a micropipetteJJ ] 2. ZD and ] "
~,.164
!
5. ICSI in human
/
5. - Lanzendoff et al. - Palermo et al.
13. SUZl in I / I humanl /
3. Laws-King I ~ ~ ' ~
I
| I
;
I
et ~l. 14, ~'Lu In I
I human I 4. Cohen et al,
Figure 1, Development of micromanipulation techniques.
Although this is not a new approach (Lanzendorf et al, 1988) (see Chapter I), it was not introduced into the routine clinical practice for several reasons.
WORLDWIDE QUESTIONNAIRE In an attempt to update knowledge on current trends in microsurgical fertilization and to focus on patient selection for the various techniques, a clinically-oriented questionnaire was designed and circulated among 53 major IVF centres around the world. Of the 53 centres, 46 responded (87%), of which 41 were actively involved in microsurgical fertilization (mean clinical experience 3.0 + 1.6 (range 0.5-7) years). Contributors are acknowledged for their data and input. The results are presented in a cumulative fashion.
)
199
WORLD SURVEY Table 1. Distribution of various microsurgical fertilization techniques practised in 41 centres in Europe, the USA, Australia and Singapore. No. of clinics practising procedure Continent
SUZI
ICSI
Europe (n = 20)
15 (> 50%)
9 (3 > 90%) 3 (30-40%)
USA (n = 14) Australia and Singapore (n = 7)
7 (:>50%) 6 (> 50%)
2 (25-45%) 3
PZD
Other
8 (4>50%) 1
1 (MIFI') 1 (LZD) 1 (MIFT) 1 (MIFT) 1 (LZD)
MIFT, microinsemination fallopian tube transfer; LZD, laser zona drilling. Percentages are out of total MF cycles performed.
The procedures described in this survey can be categorized in two main groups: (1) sperm injection techniques, i.e. SUZI, ICSI and microinjection fallopian transfer (MIFT); and (2) zona breaching techniques, i.e. PZD (mechanical) and laser zona drilling (LZD). Microdrop insemination was suggested as an alternative technique by 13 programmes. The distribution of the various procedures practised per continent is summarized in Table 1. Comments concerning miscarriages were difficult to interpret. Preliminary data suggested that miscarriage rates following microsurgical fertilization are similar to those following conventional IVF, or are somewhat higher. Patient selection
Twenty-eight centres rely mainly on total motile count (TMC) (Fishel et al, 1992; see Chapter 4) in their selection of patients for microsurgical fertilization. Seven programmes pay special attention to the morphology and few Table 2. Sperm quality selected for microsurgical fertilization.
No. of clinics reporting sperm selection Sperm quality score
TMC
1 2 3 4 5 6 7
< < < < < < >
5000 200 000 0.5 million 1 million 5 million I0 million 10 million
Did not specify No. of clinics practising procedure# Comments Centres reporting pregnancies Centres reporting live births
ICSI
SUZI
PZD
MIFT
LZD
MI
1* 3 4 1 3 1 3
1* 8 1 4 5 4 1 13
-2 3 4 2 4 -2
16
37
17
3
2
13
45 1§ 7 3
33 22
3~ 21 19
1 1
1 1
2 2
TMC, total motile count; MI, microdrop insemination. *No lower limit; tstarted recently; 3:stopped; §waiting for institutional review board.
200
Y. TADIR
rely on Kruger's criteria (Kruger et al, 1988), hemizona assay or the hamster test. The lowest TMC selected by each centre for the various procedures was categorized and scored. Table 2 summarizes the sperm-quality score, the number of clinics reporting sperm quality acceptable for each procedure, the number of clinics that practise each technique and the number of centres that reported clinical pregnancies and live births with each technique. There is a clear distinction between sperm quality selected for ICSI, SUZI and PZD. Poor sperm quality is usually selected for ICSI and SUZI. PZD is practised mainly in the presence of better sperm quality, or in patients who have failed previous IVF attempts in the presence of normal or borderline sperm quality. Two centres reported no lower limit for sperm count acceptable for microfertilization, and that a single sperm in the ejaculate may be sufficient for fertilization.
Pregnancies and babies born The questionnaire emphasized that only microsurgically induced embryos should be included in this registry. This is to avoid inaccurate conclusions concerning trends and results caused by 'mixed embryo transfers' (conventional IVF and microsurgical techniques). Accumulated data from the 41 centres revealed that 971 pregnancies were established following various types of microfertilization procedure and 426 babies were born (Table 3). Most pregnancies (833) and babies born (332) resulted from the various sperm injection techniques.
Malformations Six major malformations were reported of the 426 babies born following microsurgical fertilization (Table 4). A total of 151 karyotypes were performed in utero following the ICSI procedure and all were normal.
Table 3. Number of pregnancies and babies born following microsurgical fertilization.
No. of pregnancies
No. of babies born
SUZI ICSI SUZI + ICSI PZD PZD + SUZI MIFT
453 353"t 15 137 1 12
228 91t 11 93 1 2
Total
971
426
'Mixed' embryo transfers (conventional IVF + microsurgical technique) were not counted. *Two centres updated 20 October 1993; tone centre reported 310 ICSI pregnancies and 88 ICS1 babies born.
201
WORLD SURVEY
Table 4. Malformations reported by 41 centres practising microsurgicalfertilization (of 426 babies born). Procedure
Comments
No malformations
Various
170 normal karyotypes* One trisomy 16
ICSI SUZI
One anecephalus One cleft lip and palate (with duplication of urinary collecting system)? One hydroanecenphalus? Twins with cleft palate?
PZD Combined SUZI + ICSI
Reported by 24 centres (who had live births) Performed in utero Miscarried Out of triplet (two normal)
ICSI ICSI
Out of triplet (two normal) Both twins
*Updated by two centres 20 October 1993; ?updated by one centre 7 October 1993.
Although these results should still be considered somewhat preliminary, they are encouraging.
Tips for beginners Fifteen team leaders responded in a similar term 'experience, experience, experience' to the question: 'What message do you have for colleagues who are in the process of setting up an MM facility?' Others stressed the importance of dedicated staff, good equipment and 'start ICSI'. Four centres (two that do not practise microsurgical fertilization) suggested: 'wait for better results'.
Envoi It is too early to predict what the impact of new micromanipulation techniques on microsurgical fertilization may be. The recent trend towards sperm injection casts a shadow on the use of drilling techniques as treatment for severe male-factor infertility. Time will tell what is the grey zone in which microsurgical fertilization and conventional non-manipulated techniques compete or complement each other. As previously stated, several programmes pay special attention to some other selection methods in addition to TMC. It is not yet clear what the predictive value of these tests is, or the criteria for patient selection. However, there is little doubt that the ejaculated specimen with few motile (or even non-motile) sperm may benefit from a micromanipulating or injection technique. The information gathered in this survey is encouraging not only because of the growing numbers of pregnancies and live births that are being established, but also the relatively small number of congenital malformations. This issue is still a source for major concern as more aggressive intervention in the process of conception takes place. Improved fertilization and pregnancy rates following microsurgical fertilization will expand the spectrum of indications for these procedures.
202
Y. TADIR
Acknowledgements This work was supported by grants NIH 2RO1 CA32248 and 5P41 RR01192, DOE DE-FG0391ER61227 and ONR N00014-91-C-0134. The following people contributed to the survey: A. Acosta, S. AI Hasani, S. Amit, R.H. Asch, J.P. Balmaceda, D. Barlow, P.N. Barri, P. Brindsen, M. Bustillo, J. Cheek, J. Cohen, I. Craft, P. Dellenbach, P. Devroi, K. Diedrich, J. Donnez, J. Dor, W. Feichtinger, B. Fisch, S. Fishel, J. Garcia, L. Gianaroli, M. Glezerman, J. Gordon, S. Gordts, L. Hambereger, G. Hartshorne, B. Hedon, J. Hennessey, C. Hi, D.L. Hill, W.J. Holmgren, V. Insler, J. Itzkovitz, I. Johnston, A.M. Junca, P. Katayama, R. Kazer, J. Mandelbaum, S. Mashiach, P.L. Matson, L. Metier, D. Mortimer, S.-C. Ng, A. Obruca, T. Ord, J. Peek, I. Pike, J.P. Ryan, J. Salat Baroux, D. Saunders, R. Schoysman, J.D. Schulman, P. Serafini, J. Stangel, E. Stehlik, A. Trounson, M. Tucker, J. Tyler, P. Vanderzwalmen, A. Van Steirteghem, L.L. Veeck, A. Veiga, Y. Verlinsky, G. Warnes, A. Wittemer, D. Yang, B. Yee, Y.K. Ting.
REFERENCES Barg PE, Wahrman MZ, Talansky BE & Gordon JW (1986) Capacitated, acrosome reacted but immotile sperm, when microinjected under the zona pellucida, will not fertilize the oocyte. Journal of Experimental Zoology 237: 365-374. Cohen J (1991) A review of clinical microsurgical fertilization. In Cohen J, Malter EH, Talansky BE & Grifo J (eds) Micromanipulation of Human Gametes and Embryos, pp 163-190. Cohen J, Alikani M, Adler A et al (1992) Microsurgical fertilization procedures: the absence of stringent criteria for patient selection. Journal of Assisted Reproduction and Genetics 9: 197-206. Fishel S, Antoniori S, Jackson P e t al (1990a) Twin birth after subzonal insemination. Lancet 335: 722-723. Fishel S, Antoniori S, Jackson P e t al (1990b) Subzonal insemination for the alleviation of infertility. Fertility and Sterility 54: 828-835. Fishel S, Antinori S, Jackson P e t al (1991) Presentation of six pregnancies established by subzonal insemination (SUZI) Human Reproduction 6: 124-130. Fishel S, Timson J, Lisi F & Rinaldi L (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertility and Sterility 57: 840-849. Gordon JW & Talansky BE (1986) Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. Journal of Experimental Zoology 239: 347-354. Kruger TF, Acosta AA, Simmons KF et al (1988) Predictive value of abnormal morphology in in vitro fertilization. Fertility and Sterility 49:112-117. Lanzendorf SE, Maloney MK, Veek LL et al (1988) A preclinical evaluation of pronuclear formation by microinjection of spermatozoa into the human oocyte. Fertility and Sterility 49: 835-842. Laws-King A, Trounson A, Sathananthan H & Kola I (1987) Fertilization of the human oocyte by microinjection of single sperm under the zona pellucida. Fertility and Sterility 48: 637642. Malter EH & Cohen J (1989) Partial zona dissection of the human oocyte: a non-traumatic method using micromanipulation to assist zona pellucida penetration. Fertility and Sterility 51: 139-148. Mann JR (1988) Full term development of mouse eggs fertilized by a spermatozoon microinjected under the zona pellucida. Biology of Reproduction 38: 1077-1083. Market CL (1983) Fertilization of mammalian eggs by sperm injection. Journal of Experimental Zoology 228: 195-201. Ng S-C, Bongso A, Ratnam SS et al (1988) Pregnancy after transfer of sperm under zona. Lancet 332:790 (letter). Palermo G, Joris H, Derde MP et al (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmatic sperm injection. Fertility and Sterility 59: 826--835. Tadir Y, Wright WH & Berns MW (1989) Cell micromanipulation with laser beams. In Capi-
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