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Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection*
FERTILITY AND STERILITY Copyright
Vol. 59, No.4, April 1993
1993 The American Fertility Society
Printed on acid-free paper in U.S.A.
Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection*
Gianpiero Palermo, M.D.t Hubert Joris, M.T.t Marie-Paule Derde, Ph.D.:j:
Michel Camus, M.D.t Paul Devroey, M.D., Ph.D.t Andre Van Steirteghem, M.D., Ph.D.t§
Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University, Brussels, Belgium
Objective: To investigate the influence of sperm characteristics on the treatment by subzonal insemination (SUZI) and intracytoplasmic sperm injection of couples with severe male infertility. Design: A retrospective analysis of 300 consecutive cycles of assisted fertilization concerning 202 infertile couples was performed. One hundred fifty-three couples underwent 362 unsuccessful IVF cycles, whereas on 49 couples IVF was not performed because of poor sperm characteristics. Setting: Procedures were performed in an institutional research environment. Patients, Participants: Couples in which the male partner was the presumed cause of repeated failure to achieve conception by IVF or in which seminal parameters were unacceptable for IVF. Interventions: Three hundred transvaginal oocyte retrievals were performed after superovulation by GnRH agonist and gonadotropins. Main Outcome Measures: After SUZI and intracytoplasmic sperm injection the following parameters were evaluated: fertilization, cleavage, pregnancy, and implantation rates in relation to the sperm parameters and the proportion of acrosome-free spermatozoa after different treatments. Results: Normal fertilization occurred in 18% of the oocytes treated by SUZI and in 44% after intracytoplasmic sperm injection. Only the treatment by electroporation showed a positive correlation with the fertilization rate. Fourteen pregnancies were obtained after SUZI, 8 pregnancies after intracytoplasmic sperm injection, and 8 pregnancies after a combination of the two procedures. A score calculated from the sperm parameters after selection correlated with the fertilization obtained after SUZI, whereas a score calculated from the parameters before sperm selection correlated with the pregnancy rate. Sperm morphology influenced the implantation rate of the embryos obtained with these two procedures. Conclusions: Intracytoplasmic sperm injection and SUZI can successfully treat couples who fail IVF or who cannot benefit from IVF. Different treatments can be applied to semen samples to increase the number of acrosome-reacted spermatozoa. The few significant relations found between sperm characteristics and the outcome of assisted fertilization cannot predict the outcome. Fertil Steril 1993;59:826-35 Key Words: Sperm characteristics, micro insemination, subzonal insemination, intracytoplasmic injection
Received October 1, 1992; revised and accepted December 11, 1992.
* Supported by the Belgian Fund for Medical Research (grants 3.0036.85 and 3.0018.92), Brussels, Belgium. t Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University. t Biomedical Statistics Department, Dutch-speaking Brussels Free University. § Reprint requests: Andre Van Steirteghem, M.D., Ph.D., Centre for Reproductive Medicine, University Hospital, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
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Extensive experimental work has been carried out in the field of micromanipulation to clarify the mechanism of fertilization. Early work toward manipulated fertilization was performed by injecting hamster spermatozoa directly into the cytoplasm of hamster oocytes (1). The same experiment was then repeated with regard to mice by injecting sperm nuclei into cytochalasin-treated mouse oocytes (2). Subzonal insertion of mouse spermatozoa was performed after treatment with A23187 or after an Fertility and Sterility
incubation time of 2 hours (3, 4). Increasing the number of acrosome-reacted spermatozoa in the sperm suspension used for subzonal insemination (SUZI) of mouse oocytes increased the fertilization rate (5). A relationship between acrosome reaction and fertilization has also been clearly demonstrated (6). The success of these techniques in fertilization, embryo development, and the achievement of live offspring in experimental animals (7) led to the clinical application of SUZI (8) and intracytoplasmic sperm injection (9) to human gametes. Further study has demonstrated that by using spermatozoa from infertile men for SUZI, fertilization and cleavage were possible without increasing the incidence of chromosomal anomalies in the embryo (10). The report of the first human pregnancies and births after SUZI (11, 12, 13) and intracytoplasmic sperm injection (14) encouraged an increase in the clinical use of these procedures for patients who had failed to achieve fertilization in vitro or for those patients with too few (or only immotile) spermatozoa to even attempt IVF. Cohen et al. (15) reported recently that sperm characteristics influence the outcome of assisted fertilization techniques. A clear influence from the frequency of abnormal spermatozoa on fertilization and cleavage after partial zona dissection has been demonstrated. This study reports on 300 consecutive cycles of assisted fertilization including a previously reported first series of 44 cycles in 43 couples and deals with two relevant issues (13). First, what is the relationship, if any, between quantitative semen parameters on the one hand, before and after selection, including the percentage of acrosome-free spermatozoa and, on the other hand, the efficiency of SUZI in terms of normal fertilization: what is the influence of the frequency of abnormal spermatozoa on embryo implantation? Second, what are the differences between SUZI and intracytoplasmic sperm injection when both methods are performed on sibling 00cytes? MATERIALS AND METHODS Patients
From October 1990 until January 1992, assisted fertilization was performed in 202 couples (300 consecutive cycles) with long-standing infertility. In most cases (n = 153) at least one previous IVF attempt with an adequate nu~ber of inseminated 00Vol. 59, No.4, April 1993
cytes (mean, 10.6) had failed because oocyte fertilization was either absent or extremely reduced. In some cases (n = 49), because of the extremely low sperm parameters (::;;500,000 progressive motile spermatozoa per mL), assisted fertilization was applied immediately. In 79 cycles all the oocytes were treated by SUZI and in 15 cycles by intracytoplasmic sperm injection; in the remaining 206 cycles the two procedures were applied to sibling oocytes. The mean age of the female and male partners was 32.1 years (range, 22 to 45) and 34.8 years (range, 25 to 58), respectively. The mean duration of infertility for couples with and without a previous history of assisted procreation was 6.1 years (range, 3 to 14) and 4.6 years (range, 2 to 11), respectively. The procedure of assisted fertilization was reviewed and approved by the Institutional Ethical Committee of the Dutch-speaking Brussels Free University Medical Campus. The procedure was thoroughly explained to these couples before a detailed consent form was signed by both partners. They were also requested to sign an agreement to have prenatal diagnosis in case of a pregnancy occurring. Follicular Stimulation
Follicular stimulation was carried out by the association of GnRH agonist buserelin acetate (Suprefact; Hoechst, Brussels, Belgium), hMG (Humegon; Organon, Oss, The Netherlands or Pergonal; Serono, Brussels, Belgium), and hCG (Pregnyl, Organon; Profasi, Serono). The details of this desensitizing GnRH -a and hMG protocol have been described previously (16). Semen Analysis and Selection of Spermatozoa
Before the start of the treatment, the spermatozoa selection procedure was carried out on at least one semen sample. During the treatment the husband was asked for two semen samples: one in the morning after hCG had been administered to his wife and the second one on the morning of oocyte retrieval. Semen samples were collected by masturbation after at least 4 days of abstinence and were allowed to liquefy for at least 20 minutes at 37°C before analysis. The methodology for semen analysis and selection has been previously described (13). Semen morphology was then assessed by spreading 5 JIL of semen or sperm suspension on alcohol-ether-cleaned slides. The slides were air dried for 3 minutes, fixed for 15 seconds in Diff-Quik (Baxter Dade AG, Dudingen, Switzerland) fixative (0.002 giL fast green Palermo et al.
Sperm parameters and assisted fertilization
827
in methyl alcohol) before starting with Diff-Quik solution 1 (1.22 giL eosin G in phosphate buffer) for 10 seconds and with Diff-Quik solution 2 (1.1 giL thiazine dye in phosphate buffer) for 5 seconds. Between the fixing step and each of the staining steps, excess solution was drained from the slides by placing the slides vertically on absorbent paper. In the prior series of 44 cycles, morphology was assessed by prestained slides (Testsimplets; Boehringer Mannheim GmbH, Mannheim, Germany). Morphology was assessed according to the strict criteria described by Kruger et al. (17). Semen was considered abnormal at sperm densities < 20 X 106 /mL or at progressive motility < 40% or when <14% of the spermatozoa revealed normal morphology. The sperm sample was washed by centrifugation at 1,800 X g for 5 minutes in modified Tyrode's medium (T6) supplemented with 30 mg/mL bovine serum albumin fraction V (A-9647; Sigma Chemical Co., St. Louis, MO). The resuspended pellet was layered on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient on three layers (90%70% -50%) and centrifuged at 300 X g for 20 minutes. Where progressive motility was >20%, the spermatozoa washed from the 90% Percoll fraction were allowed to swim-up in 0.5 to 1.0 mL T6 medium. A Percoll gradient in two layers was used (95%-47.5%) (13) where samples had a sperm density < 5 X 106 /mL and <20% progressive motile spermatozoa. The Percoll fraction containing the spermatozoa was washed by adding 4 mL of T6 medium and centrifuged at 1,800 X g for 5 minutes to remove the silica gel particles. Concentration and motility of the selected spermatozoa were assessed in a Neubauer hemocytometer chamber. Enhancement and Assessment of the Acrosomal Loss
Sperm selected after Percoll was treated to enhance acrosomal loss by incubation for 24 hours in medium with follicular fluid (FF) or by electroporation. The details of these procedures have been reported previously (13). Where not enough motile spermatozoa were harvested from the semen sample incubated overnight, the semen sample collected on the day of the ovum pick-up was processed and treated by electroporation. Where <500,000 spermatozoa were present in the ejaculate, the sperm suspension was concentrated in approximately 5 p,L and transferred directly into the injection dish. The assessment of acrosomal status was performed by concanavalin A lectin and Hoechst 33258 828
Palermo et al.
Sperm parameters and assisted fertilization
(13, 18). Where the assessment of acrosomal status was performed on the sperm left after the injection procedure, approximately 20 p,L of concentrated sperm suspension was aspirated and diluted with T6 medium up to 100 p,L. After the addition of H258 and fixative, the sperm suspension was filtered through a 13-mm-diameter filter in polycarbonate with 0.6-p,m pore size (19). After addition of glycine and fluorescein isothiocyanate-conjugated concanavalin A, the filter was removed from the holder, layered on a microscope slide, and mounted for observation. Oocyte Recovery and Preparation
The technique of oocyte retrieval and classification of oocyte-cumulus complexes have been previously reported (13, 20). The cumulus-corona cells were initially removed by exposure to M2 medium containing 1 mg/mL (i.e., approximately 760 IU/mL) of hyaluronidase (Type IV -S; Sigma Chemical Co.). Subsequently, the amount of hyaluronidase was reduced to 160 IU/mL (Type VIII; Sigma Chemical Co.) Subzonal Insemination and Intracytoplasmic Sperm Injection
The preparation of the holding and injection micropipettes has already been described (13,14). The procedure was carried out on the heated stage of a Nikon Diaphot (Nikon, Tokyo, Japan) inverted microscope at 400X magnification using Nomarski optics. This microscope was equipped with two motor-driven coarse control manipulators and two hydraulic micromanipulators (M~A-188 and MO-188; Narishige Co. Ltd., Tokyo, Japan). The micropipettes were fitted to a tool holder controlled by two IM-6 microinjectors (Narishige Co. Ltd.). For the SUZI procedure, the spermatozoa were selected from the central droplet. Spermatozoa with apparently normal morphology and with slow progressive motility in the viscous environment were aspirated, tail first, into the tip of the micro injection pipette. Then the dish was moved to visualize the oocyte in an adjacent drop. The spike, if too long or too sharp, was broken against the holding pipette. This expedient provides a limited sharp area on the perimeter of the tip of the injection pipette. The oocyte was held by negative pressure on the holding pipette, and the injection needle was pushed across the zona pellucida into the perivitelline space in a region far from the polar body where the perivitelline space is narrower. A mean of three spermatozoa was Fertility and Sterility
delivered into the perivitelline space together with the smallest possible amount of medium. The pipette was then gently withdrawn, and the procedure was repeated until all oocytes had been injected. The average time required to micro inject one oocyte was approximately 1 minute. In the intracytoplasmic sperm injection procedure, the injection pipette used had an outer diameter not larger than 6 Jlm with a short, sharp spike. The spermatozoon chosen from the polyvinylpyrrolidone (PVP, PVP-K 90; Biochemicals, Cleveland, OH) drop had apparently normal morphology and was immotile. When only motile spermatozoa were present, the sperm cell chosen was immobilized by touching it with the injection needle. In cases of extreme oligospermia, the spermatozoon was aspirated from the concentrated 5 JlL of sperm suspension and transferred into the central drop where PVP solution is present to remove debris and to facilitate handling. The injection needle was introduced through the ZP and the oolemma deeply into the cytoplasm without any hesitation. The spermatozoon was injected with the smallest amount of medium possible. The pipette was slowly withdrawn to avoid removal of the sperm and eversion of the oolemma. Embryology
The handling of the oocytes immediately after the injection procedure and the evaluation of fertilization and further cleavage was performed according to our previous reports (13, 20). Oocytes were observed 16 to 18 hours after the injection procedure; survival and the number and size of pronuclei were noted. Twenty-four hours later, cleavage of the fertilized oocytes was assessed. A few hours later, up to three of the morphologically best embryos were transferred into the uterine cavity and supernumerary embryos were cryopreserved. Luteal Supplementation and Follow-up of the Pregnant Patients
The luteal phase was supported by administering 200 mg of micronized P intravaginally three times per day (21). Endocrine follow-up included serum measurements of P, 17{3-E2' and hCG-{3 at 3-day intervals until 6 weeks from the last menstrual period and once a week until 16 weeks' gestation. An ultrasound evaluation is always performed in our center to check the presence of a viable fetus in utero at 7 weeks. Biochemical pregnancy was defined as a significant increase in hCG levels (>10 mlUjmL) Vol. 59, No.4, April 1993
between days 10 and 20 after LH surge. Delayed menstruation in itself was not considered a pregnancy criterion. Clinical pregnancy implied the observation of a gestational sac at echographic screening. Ongoing pregnancy was defined when normal endocrine and echographic parameters continued to evolve beyond 20 weeks. Prenatal diagnoses were done by amnioncentesis at 16 weeks of gestation. A prospective follow-up of the children born after assisted conception is performed. Statistical Methods
All statistical tests were performed using Statistical Programs for the Social Sciences (SPSSjPC+; SPSS Inc., Chicago, IL) and Statview 512+ (BrianPower Inc., Calabasas, CA) computer programs. All statistical tests were carried out twotailed at the 5% level of significance. Percentages of oocytes fertilized with spermatozoa treated with different inducers, percentages of oocytes surviving, and percentages normally fertilized were compared for the SUZI and intracytoplasmic sperm injection procedures by X2 test. For SUZI and intracytoplasmic sperm injection the relation between embryo implantation and sperm morphology was analyzed using the MantelHaentszel test. The Pearson correlation coefficient was calculated to compare the sperm parameters, the sperm score before and after selection, and the fertilization rate. The significance of the correlation coefficient was evaluated using the t-test for correlation coefficients. The Mann-Whitney test was used to compare the sperm score of pregnant and nonpregnant patients.
RESULTS
One hundred fifty-three couples had been previously treated by IVF. Fertilization in 362 IVF treatment cycles was low: only 285 of 3,813 (7.5%) inseminated preovulatory oocytes had become fertilized. Of these couples, 88 achieved no fertilization at all. The mean values of the sperm parameters of these couples were as follows: 35.0 X 106 jmL concentration, 31.7% progressive motility, and 17.6% normal forms. Only 39 male partners had normal semen characteristics. The abnormal fertilization was assumed to be due to the impaired semen characteristics. Forty-nine couples could not be treated by IVF because of the poor semen parameters (mean values of 5.5 X 106 jmL concentration, 15.5% progressive motility, and 5.7% normal forms). Palermo et al.
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829
Table 1 Oocytes Fertilized After the Injection of Spermatozoa by One of the Two Assisted Fertilization Procedures No.ofMII oocytes Injected Survived Fertilized 2PN IPN :2:3PN
SUZI
Intracytoplasmic sperm injection
2,214 2,011 (91)*
716 548 (77) *
357 (18)t 115 39
240 (44)t 91 9
is clearly in favor of the SUZI technique (P < 0.001), whereas the number of normally fertilized oocytes is higher after the application of the intracytoplasmic sperm injection technique (357/2,214 versus 240/716; P = 0.0001). The number of oocytes with a single pronucleus was also different (P = 0.0001) in the SUZI (115/2,011) and intracytoplasmic sperm injection procedures (91/548). The percentage of three (or more) pronuclei is similar in the two procedures. We have to take into account that after intracytoplasmic sperm injection the oocytes with three pronuclei presented only one polar body. The treatment of the spermatozoa to induce acrosomalloss was performed to enhance fertilization after SUZI. Spermatozoa injected by intracytoplasmic sperm injection were treated in a similar way. The bipronucleated oocytes observed after the two injection procedures, grouped by the different sperm treatments, are shown in Table 2. In the SUZI procedure, similar fertilization rates were obtained after each of the three procedures used to induce the acrosome reaction. Only one of 48 injected 00cytes became fertilized as a result of SUZI without any treatment of the sperm suspension. From the data in the table, the treatment of the spermatozoa by electroporation after 24 hours' incubation appears to be the most successful for intracytoplasmic sperm injection. Table 3 summarizes the number of embryos transferred, the number of replacement procedures, and the number of pregnancies obtained. The data are grouped by the different assisted fertilization techniques and by the different treatments applied to the injected spermatozoa. Although in the cohort of embryos obtained after SUZI and SUZI and intracytoplasmic sperm injection, most pregnancies occurred in the group in which spermatozoa were
* Values in parentheses are percents. X2, 2 X 2, 1 df; survived versus damaged, P < 0.001. t X2, 2 X 2, 1 df; 2PN versus not fertilized/abnormally fertilized, P < 0.001.
All but 14 of 3,900 oocyte-cumulus-cell complexes were recorded as mature at the time of oocyte retrieval. After removal of the surrounding cumulus and corona cells, 3,684 oocytes (95%) revealed an intact ZP and clear cytoplasm. Eighty percent (2,945 of 3,684) had extruded the first polar body, whereas 10.5% (386 of 3,684) presented a germinal vesicle, and 9.6% (353 of 3,684) had undergone germinal vesicle breakdown. The oocytes in metaphase I stage were again evaluated before micro injection after approximately 4 hours' further incubation; 148 had then reached the metaphase II stage and were also injected. This implies that 3,093 oocytes (84%) were available for the injection procedure after approximately 4 hours' culture. The number of metaphase II oocytes that survived and became fertilized after SUZI and intracytoplasmic sperm injection procedures is described in Table 1. During SUZI, a spermatozoon was accidentally injected into the cytoplasm of 163 metaphase II oocytes. The number of oocytes surviving
Table 2
Oocytes Fertilized by One of the Two Injection Procedures in Relation to Sperm Treatment Sperm Incubation
Fertilized oocytes/injected oocytes Treatment
SUZI
Intracytoplasmic sperm injection
h
1 2 3 4 Total
1 to 4 24 1 to 4 24
FF Electroporation Electroporation
1/48 167/876 70/392 119/695 357/2,011
* Values in parentheses are percents. t 1 to 4: X2, 2 X 4, 3 df; differences within sperm treatments on fertilization after SUZI, P = 0.024. + 1 to 4: X2, 2 X 4, 3 df; differences within sperm treatments on fertilization after intracytoplasmic sperm injection, P < 0.001.
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Palermo et al.
Sperm parameters and assisted fertilization
(2)*t (19)t (I8)t (17)t (18)
21/67 33/81 84/221 102/179 240/548
(31)+ (41)+ (38)+ (57)+§ (44)
§ 4 versus 1 to 3 pooled: X2, 2 X 3, 2 df; differences between sperm treatments on fertilization after intracytoplasmic sperm injection, P < 0.001.
Fertility and Sterility
Table 3
Embryos Transferred in Terms of Different Injection Procedures and Different Sperm Treatments Sperm Procedure
Incubation
Treatment
No. of embryos
No. of transfers
85 31 54 9 8 35 42 3 42 25 30
49 20 31 5 7 21 26 2 19
No. of pregnancies
h
SUZI
Intracytoplasmic sperm injection
SUZI and intracytoplasmic sperm injection
1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24
FF Electroporation Electroporation FF Electroporation Electroporation FF Electroporation Electroporation
11
13
9 3 2 1 0 3 4 0 5 1 2
(4)* (3) (2) (1) (1)
(3) (4) (0) (2)
* Values in parentheses are percents of ongoing or to term pregnancies.
exposed to FF, most pregnancies in the group of embryos obtained after intracytoplasmic sperm injection appeared after electroporation. The relation between sperm parameters and the outcome of assisted fertilization was investigated. None of the single sperm parameters such as concentration, progressive motility, or morphology correlated with the outcome of assisted fertilization. A semen score was calculated on the basis of the number of progressive motile spermatozoa with normal morphology present in the ejaculate: volume (mL) X concentration (X106 /mL) X progressive motility (%) X normal forms (%) X 10- 4 • For the score as applied to the values observed after sperm selection, the motility parameter also included the nonprogressive forms (i.e., total motility). The sperm score calculated from the initial semen parameters correlated with the pregnancy rate (PR) (Mann-Whitney test, P = 0.036). A relationship was also found between the score calculated from the parameters' values observed after sperm selection and the fertilization rate (r = 0.21; P = 0.018). In 45 cycles it was possible to assess the acrosomal status of spermatozoa obtained from a 20-JIL sperm suspension from which sperm cells were taken for the injection procedure. Twelve semen samples were exposed for 24 hours to FF, and the mean acrosome reaction rate of the living spermatozoa was 13.9% (range, 4 to 23). In 22 semen samples incubated for 24 hours and exposed to an electric field, the mean value was 18.0% (range, 4 to 44), and in 11 cycles in which the sperm suspension was incubated for up to 4 hours and then processed by electroporation, the percentage of acrosome-free spermatozoa was 12.9% (range, 3 to 55). Only in the protocol in which Vol. 59, No.4, April 1993
electroporation was applied after 24 hours' incubation did the acrosome reaction rate correlate with the fertilization rate after SUZI (r = 0.50; P = 0.018). With the same treatment the score real acrosome reacted (%) X concentration after sperm selection (X106 /mL) X 10- 2 was clearly linked to the fertilization rate (r = 0.64; P = 0.001) obtained after SUZI. Thirty patients became pregnant: 8 pregnancies were preclinical, one clinical abortion occurred at 15 weeks of gestation because of chorion amnionitis, and 1 patient had an ectopic pregnancy requiring a salpingectomy at 7 weeks of gestation. The ongoing and to term PR was 6.7% per started cycle (20/300) and 9.8% per replacement (20/204). There were eight twin gestations (2 of which were vanishing triplets and 1 monovular twin) and 12 singleton pregnancies. In 13 patients, amniocentesis was carried out, and the fetal karyotype of 18 fetuses was normal. Seven patients decided not to have prenatal diagnosis. At the time of writing, 15 patients have delivered 21 babies (6 twin gestations), and 5 pregnancies are ongoing (2 twin). One patient delivered by cesarean section at 38 weeks because of preeclampsia. The child presented dysmaturity and cheilopalatoschisis at birth. From the 21 children born, 4 developed after the replacement of intracytoplasmic sperm injection embryos, 8 after the replacement of SUZI embryos, and 9 after SUZI and intracytoplasmic sperm injection embryos. We examined the ability to implant 294 embryos obtained after assisted fertilization by fertilization with sperm using teratozoospermic samples. Embryos (n = 120) obtained from men with extreme teratozoospermia (::S;4% normal forms) implanted at Palermo et al.
Sperm parameters and assisted fertilization
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Table 4 Correlation Between Sperm Morphology and Implantation Ability of Embryos Obtained by SUZI and Intracytoplasmic Sperm Injection Percent of normal forms
o to 4
5 to 14
No. of sacs/no. of embryos transferred Technique SUZI* Intracytoplasmic sperm injection * Total
1/43 (2.3)*
lS/134 (13.4)
4/77 (5.2) 5/120 (4.2)t
6/40 (15.0) 24/174 (13.S) t
* Mantel-Haenszel test: difference between the two techniques was not significant; values in parentheses are percents. t Difference between embryo implantation related to sperm morphology, controlled for the technique used, P = 0.007.
a significantly lower rate than embryos (n = 174) from men with less severe teratozoospermia (5% to 14% normal forms). As indicated in Table 4, there was no difference between the SUZI and intracytoplasmic sperm injection procedures. Table 5 summarizes the relationship between sperm characteristics, previous history of assisted reproduction, and fertilization obtained after SUZI and intracytoplasmic sperm injection procedures, including the pregnancies. In all classes of sperm
density, the fertilization rate obtained after the intracytoplasmic sperm injection procedure is clearly better than after the SUZI. Surprisingly, the class 20 to 40 X 106 /mL has a lower fertilization rate using the intracytoplasmic sperm injection procedure than with the others. Twelve pregnancies (only 5 ongoing and to term) appear in the group with sperm density ~ 40 X 106 /mL. Table 6 reports the results of assisted fertilization related to the sperm characteristics in couples without previous IVF because of too poor sperm values. In this series, too, the intracytoplasmic sperm injection-treated oocytes appear to fertilize better than the SUZI-treated, except for the category with the highest density (class> 20 X 106 /mL). The incidence of pregnancies in this group is lower than among the patients with a previous IVF history, whereas the fertilization rate obtained after each of the two injection procedures is similar. DISCUSSION
In this study, 202 couples were treated in 300 consecutive cycles of assisted fertilization. Subzonal insemination was performed in 2,214 oocytes, of which 357 fertilized normally. A new procedure, the intracytoplasmic sperm injection of a single spermatozoon, was performed on 716 oocytes, 240 of which fertilized normally.
Table 5 Fertilization and Pregnancies After Assisted Fertilization Related to Sperm Characteristics in Patients With Previous History of Assisted Reproduction Classes of sperm density*
Sperm characteristics Concentration (X106/mL) Progressive motility (%) Normal forms (%) Outcome of assisted reproduction No. of previous IVF cycles No. of zygotes after IVF/no. of oocytes Outcome of assisted fertilization No. of cycles No. of zygotes/no. of oocytes injected by SUZI No. of zygotes/no. of oocytes after intracytoplasmic sperm injection No. of pregnancies/no. of transfers No. of pregnancies ongoing or to term
,,;1
1 to 5
5 to 20
20 to 40
;0>40
0.5 (0 to 1) t 27.4 (0 to Sl) 3.5 (0 to 12)
3.1 (1.2 to 5) 19.5 (0 to 50) II (0 to 43)
11.7 (5.1 to 20) 26.3 (0 to SO) 14.0 (0 to 47)
30.0 (20.2 to 40) 34.7 (0 to SO) 20.2 (0 to 50)
S1.5 (40.6 to 200) 41.2 (2 to SO) 25.5 (0 to 6S)
14
64
(1 to 3)
6/133 [4.5]:j:
(1 to 6)
35/676 [5.2]
19 7/94
[7.4]
32/74 [43.2] 4/14
Palermo et al.
100/1,032 [9.7]
69
(1 to 6)
32/749 [4.3]
63
llO
(1 to 9)
112/1,223 [9.2] 75
37
30/2S6 [10.5]
64/396
[16.2]
63/296 [21.3]
131/593
[22.1]
26/50 [52.0]
30/63
[47.6]
19/57 [33.3]
42/72
[5S.3]
6/26
3/3S 2
* Density X 106 /mL. t Values are means with ranges in parentheses.
832
(1 to 6)
37
2/21 3
105
Sperm parameters and assisted fertilization
3
12/52 5
5
:j: Values in brackets are percents of fertilization.
Fertility and Sterility
Table 6
Fertilization and Pregnancies After Assisted Fertilization in Patients With Very Poor Semen Characteristics Classes of sperm density'
Sperm characteristics Concentration (X10 6 /mL) Progressive motility (%) Normal forms (%) Epididymal sperm Outcome of assisted fertilization No. of cycles No. of zygotes/no. of oocytes injected by SUZI No. of zygotes/no. of oocytes after intracytoplasmic sperm injection No. of pregnancies/no. of transfers No. of pregnancies ongoing or to term
.,;1
1 to 5
5 to 20
>20
0.3 (0 to l)t 17.0 (0 to 70) 3.5 (0 to 20) One cycle
2.7 (1.1 to 4.9) 15.5 (0 to 63) 4.6 (0 to 24)
8.5 (4.9 to 12.8) 16.8 (0 to 62) 5.9 (0 to 22)
44.8 (34.5 to 58) 2.3 (0 to 6) 23.0 (20 to 28)
29
19
16
4
10/110 [9.1]:j:
23/127 [18.1]
18/77 [23.4]
11/32 [34.4]
55/150 [36.7]
25/56 [44.6]
10/23 [43.5]
1/3 [33.3]
1/22
2/17 1
• Density X 106/mL. t Values are means with ranges in parentheses.
Subzonal insemination can lead to fertilization in previous IVF failures and in cases of extremely impaired semen parameters (8, 15, 22, 23). Furthermore, the SUZI procedure does not result in a significant increase in abnormal embryos (10) or in the birth of abnormal babies (12, 13, 24). In a previous report we have also shown that patients treated by intracytoplasmic sperm injection can achieve pregnancy and give birth to normal babies (14). The main difference between the two techniques is that SUZI requires few but functional spermatozoa, whereas in the intracytoplasmic sperm injection technique, acrosomeless spermatozoa may also be used. Furthermore, in the intracytoplasmic sperm injection technique a single spermatozoon is injected. We observed a better (P = 0.024) fertilization rate after the injection of spermatozoa treated to induce the acrosome reaction than with oocytes injected with untreated spermatozoa (Table 2). Assessment of the acrosomal status of spermatozoa exposed to different treatments showed a mean acrosome reaction rate of 14.9%. In 22 observations in which spermatozoa were treated by an electric field (1,250 Vfcm for 2.5 ms and 24 hours' incubation) and injected subzonally, the oocytes were fertilized in relation to the proportion of acrosome-reacted spermatozoa. In a previous report by our group, a positive correlation was found between the proportion of acrosome-reacted spermatozoa in the sperm suspension and the number of fertilized oocytes obtained after the subzonal insertion of a single mouse spermatozoon (6). Similarly, the cohort of oocytes Vol. 59, No.4, April 1993
0/11
0/3
1 :j: Values in brackets are percents of fertilization.
treated by intracytoplasmic sperm injection showed differences in fertilization. In fact, after one treatment, i.e., electroporation after 24 hours' incubation, the fertilization rate was remarkable. In the oocytes on which intracytoplasmic sperm injection was carried out, untreated spermatozoa fertilized at a rate similar to that of treated spermatozoa. This observation seems to indicate that the treatments applied to induce acrosomal loss affect not only the acrosomal status. Analyzing these data, we were not able to find a clear relationship between a single impaired sperm parameter and the number of oocytes fertilized, the number and quality of the embryos, and the number of pregnancies obtained. By calculating a score taking into account the total number of progressive motile spermatozoa and the frequency of morphologically normal spermatozoa in the ejaculates, we were able to observe a relationship with the number of pregnancies. When the semen score included the sperm values after sperm selection, a correlation with the number of oocytes fertilized by SUZI was found. Furthermore, embryos obtained after the SUZI or intracytoplasmic sperm injection were able to implant at a different rate in relation to the different incidence of morphologically abnormal spermatozoa present in the semen sample. This demonstrates that embryos derived from microsurgical fertilization with extreme teratozoospermia «5% normal forms) were severely impaired (P = 0.0138) in their ability to implant irrespective of whether they were treated by SUZI or intracytoplasmic Palermo et al.
Sperm parameters and assisted fertilization
833
sperm injection. These relative differences in implantation of embryos fertilized with semen with severe teratozoospermia require confirmation on a larger number of pregnancies. These findings are in disagreement with Cohen et al. (15) who claim that the influence of the morphological sperm factor differs in the different microsurgical fertilization procedures used. These assisted fertilization procedures can successfully treat couples who do not conceive after classical IVF or couples with very poor semen parameters. However, some limitations have to be taken into consideration. The enzymatic and mechanical removal of the cumulus-corona cells from the oocytes necessary to allow their manipulation and visualization can be deleterious. This exposure of oocytes to hyaluronidase can induce parthenogenetic activation (23). This phenomenon appears to be more frequent than in the classic IVF (25). The injection procedures per se can damage the oocyte and may also induce parthenogenetic activation. The intracytoplasmic sperm injection procedure can cause more frequent injuries to the oocyte than the SUZI, but the higher fertilization rate after intracytoplasmic sperm injection compensates for this. Although with the intracytoplasmic sperm injection technique we overcome in theory the problem of multiple sperm penetration that besets partial zona dissection and at times SUZI, we sometimes observe the presence of three pronuclei oocytes in the intracytoplasmic sperm injection-treated oocytes; this may be due to nonextrusion of the second polar body. Similarly, embryo cleavage and quality can be influenced by these procedures. This becomes particularly so when very poor semen is used (e.g., in the intracytoplasmic sperm injection) and may also influence the implantation of embryos derived from the injection of poor semen. Although previous reports of our experiences of SUZI and intracytoplasmic sperm injection (13, 14) are encouraging, the evolution of pregnancies obtained by microinjection has to be carefully assessed. At the time of writing, 20 pregnancies have developed to term or are still ongoing. Because few children are born after assisted fertilization, a strict monitoring of the pregnancies by the study of the fetal karyotype and pediatric follow-up ofthe children born is advisable. Acknowledgments. We thank the clinical and scientific staff of the Centre for Reproductive Medicine, Brussels, Belgium, for their expert assistance; Ms. Geertrui Bocken and Ms. An Vankelecom for technical assistance; Ms. Annick Geril and colleagues for counseling; Mr. Frank Winter of the Language Education
834
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Sperm parameters and assisted fertilization
Center for proofreading the text in English; Inge Liebaers, M.D., Ph.D., Department of Medical Genetics of the Dutch-speaking Brussels Free University, Brussels, Belgium, for the genetic counseling and for analyzing the fetal karyotypes; and Ms. Viviane De Wolf for typing the manuscript.
REFERENCES 1. Uehara T, Yanagimachi R. Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Bioi Reprod 1976;15: 467-70. 2. Markert CL. Fertilization of mammalian eggs by sperm injection. J Exp Zool 1983;228:195-201. 3. Barg PE, Wahrman MZ, Talansky BE, Gordon JW. Capacitated, acrosome reacted but immotile sperm, when microinjected under the mouse zona pellucida, will not fertilize the oocyte. J Exp Zoo I 1986;237:365-74. 4. Mann JR. Full term development of mouse eggs fertilized by a spermatozoon microinjected under the zona pellucida. Bioi Reprod 1988;38:1077-83. 5. Lacham 0, Trounson A, Holden C, Mann J, Sathananthan H. Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induce the acrosome reaction. Gamete Res 1989;23:233-4. 6. Palermo G, Van Steirteghem AC. Enhancement of acrosome reaction and subzonal insemination of a single spermatozoon in mouse eggs. Mol Reprod Dev 1991;30:339-345. 7. Iritani A. Micromanipulation of gametes for in vitro assisted fertilization. Mol Reprod Dev 1991;28:199-207. 8. Laws-King A, Trounson A, Sathananthan H, Kola I. Fertilization of human oocytes by micro injection of a single spermatozoon under the zona pellucida. Fertil SteriI1987;48:63742. 9. Lanzendorf SE, Maloney MK, Veeck LL, Slusser J, Hodgen GD, Rosenwaks Z. A preclinical evaluation of pronuclear formation by micro injection of human spermatozoa into human oocytes. Fertil Steril 1988;49:835-42. 10. Kola I, Lacham 0, Jansen RPS, Turner M, Trounson A. Chromosomal analysis of human oocytes fertilized by microinjection of spermatozoa into the perivitelline space. Hum Reprod 1990;5:575-7. 11. Ng S-C, Bongso A, Ratnam SS, Sathananthan H, Chan CLK, Wong PC, et al. Pregnancy after transfer of sperm under zona [letter]. Lancet 1988;332:790. 12. Fishel S, Antinori S, Jackson P, Johnson J, Lisi F, Chiariello F, et al. Twin birth after sub-zonal insemination. Lancet 1990;335:722-3. 13. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Induction of acrosome reaction in human spermatozoa used for subzonal insemination. Hum Reprod 1992;7:248-54. 14. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-8. 15. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, Reing A, et al. Microsurgical fertilization and teratozoospermia. Hum Reprod 1991;6:118-23. 16. Smitz J, Devroey P, Braeckmans P, Camus M, Khan I, Staessen C, et al. Management of failed cycles in an IVF / GIFT programme with the combination of a GnRH analogue and hMG. Hum Reprod 1987;2:309-14. 17. Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphologic features
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18.
19.
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as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46:1118-23. Holden CA, Hyne RV, Sathananthan AH, Trounson AO. Assessment of the human sperm acrosome reaction using concanavalin A lectin. Mol Reprod Dev 1990;25:247-57. Morales P, Cross NL. A new procedure for determining acrosomal status of very small numbers of human sperm. J Histochem Cytochem 1989;37:1291-2. Staessen C, Camus M, Khan I, Smitz J, Van Waesberghe L, Wisanto A, et al. An 18-month survey of infertility treatment by in vitro fertilization, gamete and zygote intrafallopian transfer, and replacement of frozen-thawed embryos. J In Vitro Fert Embryo Transf 1989;6:22-9. Smitz J, Devroey P, Faguer B, Bourgain C, Camus M, Van Steirteghem AC. A prospective randomized comparison of
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22.
23.
24.
25.
intramuscular or intravaginal natural progesterone as a luteal phase and early pregnancy supplement. Hum Reprod 1992;7: 168-75. Bongso TA, Sathananthan AH, Wong PC, Ratnam SS, Ng SC, Anandakumar C, et al. Human fertilization by micro-injection of immotile spermatozoa. Hum Reprod 1989;4:175-9. Fishel S, Timson J, Lisi F, Rinaldi L. Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertil Steril1992;57:840-9. Fishel S, Antinori S, Jackson P, Johnson J, Rinaldi L. Presentation of six pregnancies established by subzonal insemination (SUZI). Hum Reprod 1991;6:124-30. Plachot M, Junca AM, Mandelbaum J, de Grouchy J, SalatBaroux J, Cohen J. Chromosome investigation in early life. II. Human preimplantation embryos. Hum Reprod 1987;2: 29-35.
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Vol. 59, No.4, April 1993
1993 The American Fertility Society
Printed on acid-free paper in U.S.A.
Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection*
Gianpiero Palermo, M.D.t Hubert Joris, M.T.t Marie-Paule Derde, Ph.D.:j:
Michel Camus, M.D.t Paul Devroey, M.D., Ph.D.t Andre Van Steirteghem, M.D., Ph.D.t§
Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University, Brussels, Belgium
Objective: To investigate the influence of sperm characteristics on the treatment by subzonal insemination (SUZI) and intracytoplasmic sperm injection of couples with severe male infertility. Design: A retrospective analysis of 300 consecutive cycles of assisted fertilization concerning 202 infertile couples was performed. One hundred fifty-three couples underwent 362 unsuccessful IVF cycles, whereas on 49 couples IVF was not performed because of poor sperm characteristics. Setting: Procedures were performed in an institutional research environment. Patients, Participants: Couples in which the male partner was the presumed cause of repeated failure to achieve conception by IVF or in which seminal parameters were unacceptable for IVF. Interventions: Three hundred transvaginal oocyte retrievals were performed after superovulation by GnRH agonist and gonadotropins. Main Outcome Measures: After SUZI and intracytoplasmic sperm injection the following parameters were evaluated: fertilization, cleavage, pregnancy, and implantation rates in relation to the sperm parameters and the proportion of acrosome-free spermatozoa after different treatments. Results: Normal fertilization occurred in 18% of the oocytes treated by SUZI and in 44% after intracytoplasmic sperm injection. Only the treatment by electroporation showed a positive correlation with the fertilization rate. Fourteen pregnancies were obtained after SUZI, 8 pregnancies after intracytoplasmic sperm injection, and 8 pregnancies after a combination of the two procedures. A score calculated from the sperm parameters after selection correlated with the fertilization obtained after SUZI, whereas a score calculated from the parameters before sperm selection correlated with the pregnancy rate. Sperm morphology influenced the implantation rate of the embryos obtained with these two procedures. Conclusions: Intracytoplasmic sperm injection and SUZI can successfully treat couples who fail IVF or who cannot benefit from IVF. Different treatments can be applied to semen samples to increase the number of acrosome-reacted spermatozoa. The few significant relations found between sperm characteristics and the outcome of assisted fertilization cannot predict the outcome. Fertil Steril 1993;59:826-35 Key Words: Sperm characteristics, micro insemination, subzonal insemination, intracytoplasmic injection
Received October 1, 1992; revised and accepted December 11, 1992.
* Supported by the Belgian Fund for Medical Research (grants 3.0036.85 and 3.0018.92), Brussels, Belgium. t Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University. t Biomedical Statistics Department, Dutch-speaking Brussels Free University. § Reprint requests: Andre Van Steirteghem, M.D., Ph.D., Centre for Reproductive Medicine, University Hospital, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
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Sperm parameters and assisted fertilization
Extensive experimental work has been carried out in the field of micromanipulation to clarify the mechanism of fertilization. Early work toward manipulated fertilization was performed by injecting hamster spermatozoa directly into the cytoplasm of hamster oocytes (1). The same experiment was then repeated with regard to mice by injecting sperm nuclei into cytochalasin-treated mouse oocytes (2). Subzonal insertion of mouse spermatozoa was performed after treatment with A23187 or after an Fertility and Sterility
incubation time of 2 hours (3, 4). Increasing the number of acrosome-reacted spermatozoa in the sperm suspension used for subzonal insemination (SUZI) of mouse oocytes increased the fertilization rate (5). A relationship between acrosome reaction and fertilization has also been clearly demonstrated (6). The success of these techniques in fertilization, embryo development, and the achievement of live offspring in experimental animals (7) led to the clinical application of SUZI (8) and intracytoplasmic sperm injection (9) to human gametes. Further study has demonstrated that by using spermatozoa from infertile men for SUZI, fertilization and cleavage were possible without increasing the incidence of chromosomal anomalies in the embryo (10). The report of the first human pregnancies and births after SUZI (11, 12, 13) and intracytoplasmic sperm injection (14) encouraged an increase in the clinical use of these procedures for patients who had failed to achieve fertilization in vitro or for those patients with too few (or only immotile) spermatozoa to even attempt IVF. Cohen et al. (15) reported recently that sperm characteristics influence the outcome of assisted fertilization techniques. A clear influence from the frequency of abnormal spermatozoa on fertilization and cleavage after partial zona dissection has been demonstrated. This study reports on 300 consecutive cycles of assisted fertilization including a previously reported first series of 44 cycles in 43 couples and deals with two relevant issues (13). First, what is the relationship, if any, between quantitative semen parameters on the one hand, before and after selection, including the percentage of acrosome-free spermatozoa and, on the other hand, the efficiency of SUZI in terms of normal fertilization: what is the influence of the frequency of abnormal spermatozoa on embryo implantation? Second, what are the differences between SUZI and intracytoplasmic sperm injection when both methods are performed on sibling 00cytes? MATERIALS AND METHODS Patients
From October 1990 until January 1992, assisted fertilization was performed in 202 couples (300 consecutive cycles) with long-standing infertility. In most cases (n = 153) at least one previous IVF attempt with an adequate nu~ber of inseminated 00Vol. 59, No.4, April 1993
cytes (mean, 10.6) had failed because oocyte fertilization was either absent or extremely reduced. In some cases (n = 49), because of the extremely low sperm parameters (::;;500,000 progressive motile spermatozoa per mL), assisted fertilization was applied immediately. In 79 cycles all the oocytes were treated by SUZI and in 15 cycles by intracytoplasmic sperm injection; in the remaining 206 cycles the two procedures were applied to sibling oocytes. The mean age of the female and male partners was 32.1 years (range, 22 to 45) and 34.8 years (range, 25 to 58), respectively. The mean duration of infertility for couples with and without a previous history of assisted procreation was 6.1 years (range, 3 to 14) and 4.6 years (range, 2 to 11), respectively. The procedure of assisted fertilization was reviewed and approved by the Institutional Ethical Committee of the Dutch-speaking Brussels Free University Medical Campus. The procedure was thoroughly explained to these couples before a detailed consent form was signed by both partners. They were also requested to sign an agreement to have prenatal diagnosis in case of a pregnancy occurring. Follicular Stimulation
Follicular stimulation was carried out by the association of GnRH agonist buserelin acetate (Suprefact; Hoechst, Brussels, Belgium), hMG (Humegon; Organon, Oss, The Netherlands or Pergonal; Serono, Brussels, Belgium), and hCG (Pregnyl, Organon; Profasi, Serono). The details of this desensitizing GnRH -a and hMG protocol have been described previously (16). Semen Analysis and Selection of Spermatozoa
Before the start of the treatment, the spermatozoa selection procedure was carried out on at least one semen sample. During the treatment the husband was asked for two semen samples: one in the morning after hCG had been administered to his wife and the second one on the morning of oocyte retrieval. Semen samples were collected by masturbation after at least 4 days of abstinence and were allowed to liquefy for at least 20 minutes at 37°C before analysis. The methodology for semen analysis and selection has been previously described (13). Semen morphology was then assessed by spreading 5 JIL of semen or sperm suspension on alcohol-ether-cleaned slides. The slides were air dried for 3 minutes, fixed for 15 seconds in Diff-Quik (Baxter Dade AG, Dudingen, Switzerland) fixative (0.002 giL fast green Palermo et al.
Sperm parameters and assisted fertilization
827
in methyl alcohol) before starting with Diff-Quik solution 1 (1.22 giL eosin G in phosphate buffer) for 10 seconds and with Diff-Quik solution 2 (1.1 giL thiazine dye in phosphate buffer) for 5 seconds. Between the fixing step and each of the staining steps, excess solution was drained from the slides by placing the slides vertically on absorbent paper. In the prior series of 44 cycles, morphology was assessed by prestained slides (Testsimplets; Boehringer Mannheim GmbH, Mannheim, Germany). Morphology was assessed according to the strict criteria described by Kruger et al. (17). Semen was considered abnormal at sperm densities < 20 X 106 /mL or at progressive motility < 40% or when <14% of the spermatozoa revealed normal morphology. The sperm sample was washed by centrifugation at 1,800 X g for 5 minutes in modified Tyrode's medium (T6) supplemented with 30 mg/mL bovine serum albumin fraction V (A-9647; Sigma Chemical Co., St. Louis, MO). The resuspended pellet was layered on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient on three layers (90%70% -50%) and centrifuged at 300 X g for 20 minutes. Where progressive motility was >20%, the spermatozoa washed from the 90% Percoll fraction were allowed to swim-up in 0.5 to 1.0 mL T6 medium. A Percoll gradient in two layers was used (95%-47.5%) (13) where samples had a sperm density < 5 X 106 /mL and <20% progressive motile spermatozoa. The Percoll fraction containing the spermatozoa was washed by adding 4 mL of T6 medium and centrifuged at 1,800 X g for 5 minutes to remove the silica gel particles. Concentration and motility of the selected spermatozoa were assessed in a Neubauer hemocytometer chamber. Enhancement and Assessment of the Acrosomal Loss
Sperm selected after Percoll was treated to enhance acrosomal loss by incubation for 24 hours in medium with follicular fluid (FF) or by electroporation. The details of these procedures have been reported previously (13). Where not enough motile spermatozoa were harvested from the semen sample incubated overnight, the semen sample collected on the day of the ovum pick-up was processed and treated by electroporation. Where <500,000 spermatozoa were present in the ejaculate, the sperm suspension was concentrated in approximately 5 p,L and transferred directly into the injection dish. The assessment of acrosomal status was performed by concanavalin A lectin and Hoechst 33258 828
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Sperm parameters and assisted fertilization
(13, 18). Where the assessment of acrosomal status was performed on the sperm left after the injection procedure, approximately 20 p,L of concentrated sperm suspension was aspirated and diluted with T6 medium up to 100 p,L. After the addition of H258 and fixative, the sperm suspension was filtered through a 13-mm-diameter filter in polycarbonate with 0.6-p,m pore size (19). After addition of glycine and fluorescein isothiocyanate-conjugated concanavalin A, the filter was removed from the holder, layered on a microscope slide, and mounted for observation. Oocyte Recovery and Preparation
The technique of oocyte retrieval and classification of oocyte-cumulus complexes have been previously reported (13, 20). The cumulus-corona cells were initially removed by exposure to M2 medium containing 1 mg/mL (i.e., approximately 760 IU/mL) of hyaluronidase (Type IV -S; Sigma Chemical Co.). Subsequently, the amount of hyaluronidase was reduced to 160 IU/mL (Type VIII; Sigma Chemical Co.) Subzonal Insemination and Intracytoplasmic Sperm Injection
The preparation of the holding and injection micropipettes has already been described (13,14). The procedure was carried out on the heated stage of a Nikon Diaphot (Nikon, Tokyo, Japan) inverted microscope at 400X magnification using Nomarski optics. This microscope was equipped with two motor-driven coarse control manipulators and two hydraulic micromanipulators (M~A-188 and MO-188; Narishige Co. Ltd., Tokyo, Japan). The micropipettes were fitted to a tool holder controlled by two IM-6 microinjectors (Narishige Co. Ltd.). For the SUZI procedure, the spermatozoa were selected from the central droplet. Spermatozoa with apparently normal morphology and with slow progressive motility in the viscous environment were aspirated, tail first, into the tip of the micro injection pipette. Then the dish was moved to visualize the oocyte in an adjacent drop. The spike, if too long or too sharp, was broken against the holding pipette. This expedient provides a limited sharp area on the perimeter of the tip of the injection pipette. The oocyte was held by negative pressure on the holding pipette, and the injection needle was pushed across the zona pellucida into the perivitelline space in a region far from the polar body where the perivitelline space is narrower. A mean of three spermatozoa was Fertility and Sterility
delivered into the perivitelline space together with the smallest possible amount of medium. The pipette was then gently withdrawn, and the procedure was repeated until all oocytes had been injected. The average time required to micro inject one oocyte was approximately 1 minute. In the intracytoplasmic sperm injection procedure, the injection pipette used had an outer diameter not larger than 6 Jlm with a short, sharp spike. The spermatozoon chosen from the polyvinylpyrrolidone (PVP, PVP-K 90; Biochemicals, Cleveland, OH) drop had apparently normal morphology and was immotile. When only motile spermatozoa were present, the sperm cell chosen was immobilized by touching it with the injection needle. In cases of extreme oligospermia, the spermatozoon was aspirated from the concentrated 5 JlL of sperm suspension and transferred into the central drop where PVP solution is present to remove debris and to facilitate handling. The injection needle was introduced through the ZP and the oolemma deeply into the cytoplasm without any hesitation. The spermatozoon was injected with the smallest amount of medium possible. The pipette was slowly withdrawn to avoid removal of the sperm and eversion of the oolemma. Embryology
The handling of the oocytes immediately after the injection procedure and the evaluation of fertilization and further cleavage was performed according to our previous reports (13, 20). Oocytes were observed 16 to 18 hours after the injection procedure; survival and the number and size of pronuclei were noted. Twenty-four hours later, cleavage of the fertilized oocytes was assessed. A few hours later, up to three of the morphologically best embryos were transferred into the uterine cavity and supernumerary embryos were cryopreserved. Luteal Supplementation and Follow-up of the Pregnant Patients
The luteal phase was supported by administering 200 mg of micronized P intravaginally three times per day (21). Endocrine follow-up included serum measurements of P, 17{3-E2' and hCG-{3 at 3-day intervals until 6 weeks from the last menstrual period and once a week until 16 weeks' gestation. An ultrasound evaluation is always performed in our center to check the presence of a viable fetus in utero at 7 weeks. Biochemical pregnancy was defined as a significant increase in hCG levels (>10 mlUjmL) Vol. 59, No.4, April 1993
between days 10 and 20 after LH surge. Delayed menstruation in itself was not considered a pregnancy criterion. Clinical pregnancy implied the observation of a gestational sac at echographic screening. Ongoing pregnancy was defined when normal endocrine and echographic parameters continued to evolve beyond 20 weeks. Prenatal diagnoses were done by amnioncentesis at 16 weeks of gestation. A prospective follow-up of the children born after assisted conception is performed. Statistical Methods
All statistical tests were performed using Statistical Programs for the Social Sciences (SPSSjPC+; SPSS Inc., Chicago, IL) and Statview 512+ (BrianPower Inc., Calabasas, CA) computer programs. All statistical tests were carried out twotailed at the 5% level of significance. Percentages of oocytes fertilized with spermatozoa treated with different inducers, percentages of oocytes surviving, and percentages normally fertilized were compared for the SUZI and intracytoplasmic sperm injection procedures by X2 test. For SUZI and intracytoplasmic sperm injection the relation between embryo implantation and sperm morphology was analyzed using the MantelHaentszel test. The Pearson correlation coefficient was calculated to compare the sperm parameters, the sperm score before and after selection, and the fertilization rate. The significance of the correlation coefficient was evaluated using the t-test for correlation coefficients. The Mann-Whitney test was used to compare the sperm score of pregnant and nonpregnant patients.
RESULTS
One hundred fifty-three couples had been previously treated by IVF. Fertilization in 362 IVF treatment cycles was low: only 285 of 3,813 (7.5%) inseminated preovulatory oocytes had become fertilized. Of these couples, 88 achieved no fertilization at all. The mean values of the sperm parameters of these couples were as follows: 35.0 X 106 jmL concentration, 31.7% progressive motility, and 17.6% normal forms. Only 39 male partners had normal semen characteristics. The abnormal fertilization was assumed to be due to the impaired semen characteristics. Forty-nine couples could not be treated by IVF because of the poor semen parameters (mean values of 5.5 X 106 jmL concentration, 15.5% progressive motility, and 5.7% normal forms). Palermo et al.
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Table 1 Oocytes Fertilized After the Injection of Spermatozoa by One of the Two Assisted Fertilization Procedures No.ofMII oocytes Injected Survived Fertilized 2PN IPN :2:3PN
SUZI
Intracytoplasmic sperm injection
2,214 2,011 (91)*
716 548 (77) *
357 (18)t 115 39
240 (44)t 91 9
is clearly in favor of the SUZI technique (P < 0.001), whereas the number of normally fertilized oocytes is higher after the application of the intracytoplasmic sperm injection technique (357/2,214 versus 240/716; P = 0.0001). The number of oocytes with a single pronucleus was also different (P = 0.0001) in the SUZI (115/2,011) and intracytoplasmic sperm injection procedures (91/548). The percentage of three (or more) pronuclei is similar in the two procedures. We have to take into account that after intracytoplasmic sperm injection the oocytes with three pronuclei presented only one polar body. The treatment of the spermatozoa to induce acrosomalloss was performed to enhance fertilization after SUZI. Spermatozoa injected by intracytoplasmic sperm injection were treated in a similar way. The bipronucleated oocytes observed after the two injection procedures, grouped by the different sperm treatments, are shown in Table 2. In the SUZI procedure, similar fertilization rates were obtained after each of the three procedures used to induce the acrosome reaction. Only one of 48 injected 00cytes became fertilized as a result of SUZI without any treatment of the sperm suspension. From the data in the table, the treatment of the spermatozoa by electroporation after 24 hours' incubation appears to be the most successful for intracytoplasmic sperm injection. Table 3 summarizes the number of embryos transferred, the number of replacement procedures, and the number of pregnancies obtained. The data are grouped by the different assisted fertilization techniques and by the different treatments applied to the injected spermatozoa. Although in the cohort of embryos obtained after SUZI and SUZI and intracytoplasmic sperm injection, most pregnancies occurred in the group in which spermatozoa were
* Values in parentheses are percents. X2, 2 X 2, 1 df; survived versus damaged, P < 0.001. t X2, 2 X 2, 1 df; 2PN versus not fertilized/abnormally fertilized, P < 0.001.
All but 14 of 3,900 oocyte-cumulus-cell complexes were recorded as mature at the time of oocyte retrieval. After removal of the surrounding cumulus and corona cells, 3,684 oocytes (95%) revealed an intact ZP and clear cytoplasm. Eighty percent (2,945 of 3,684) had extruded the first polar body, whereas 10.5% (386 of 3,684) presented a germinal vesicle, and 9.6% (353 of 3,684) had undergone germinal vesicle breakdown. The oocytes in metaphase I stage were again evaluated before micro injection after approximately 4 hours' further incubation; 148 had then reached the metaphase II stage and were also injected. This implies that 3,093 oocytes (84%) were available for the injection procedure after approximately 4 hours' culture. The number of metaphase II oocytes that survived and became fertilized after SUZI and intracytoplasmic sperm injection procedures is described in Table 1. During SUZI, a spermatozoon was accidentally injected into the cytoplasm of 163 metaphase II oocytes. The number of oocytes surviving
Table 2
Oocytes Fertilized by One of the Two Injection Procedures in Relation to Sperm Treatment Sperm Incubation
Fertilized oocytes/injected oocytes Treatment
SUZI
Intracytoplasmic sperm injection
h
1 2 3 4 Total
1 to 4 24 1 to 4 24
FF Electroporation Electroporation
1/48 167/876 70/392 119/695 357/2,011
* Values in parentheses are percents. t 1 to 4: X2, 2 X 4, 3 df; differences within sperm treatments on fertilization after SUZI, P = 0.024. + 1 to 4: X2, 2 X 4, 3 df; differences within sperm treatments on fertilization after intracytoplasmic sperm injection, P < 0.001.
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Palermo et al.
Sperm parameters and assisted fertilization
(2)*t (19)t (I8)t (17)t (18)
21/67 33/81 84/221 102/179 240/548
(31)+ (41)+ (38)+ (57)+§ (44)
§ 4 versus 1 to 3 pooled: X2, 2 X 3, 2 df; differences between sperm treatments on fertilization after intracytoplasmic sperm injection, P < 0.001.
Fertility and Sterility
Table 3
Embryos Transferred in Terms of Different Injection Procedures and Different Sperm Treatments Sperm Procedure
Incubation
Treatment
No. of embryos
No. of transfers
85 31 54 9 8 35 42 3 42 25 30
49 20 31 5 7 21 26 2 19
No. of pregnancies
h
SUZI
Intracytoplasmic sperm injection
SUZI and intracytoplasmic sperm injection
1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24 1 to 4 24
FF Electroporation Electroporation FF Electroporation Electroporation FF Electroporation Electroporation
11
13
9 3 2 1 0 3 4 0 5 1 2
(4)* (3) (2) (1) (1)
(3) (4) (0) (2)
* Values in parentheses are percents of ongoing or to term pregnancies.
exposed to FF, most pregnancies in the group of embryos obtained after intracytoplasmic sperm injection appeared after electroporation. The relation between sperm parameters and the outcome of assisted fertilization was investigated. None of the single sperm parameters such as concentration, progressive motility, or morphology correlated with the outcome of assisted fertilization. A semen score was calculated on the basis of the number of progressive motile spermatozoa with normal morphology present in the ejaculate: volume (mL) X concentration (X106 /mL) X progressive motility (%) X normal forms (%) X 10- 4 • For the score as applied to the values observed after sperm selection, the motility parameter also included the nonprogressive forms (i.e., total motility). The sperm score calculated from the initial semen parameters correlated with the pregnancy rate (PR) (Mann-Whitney test, P = 0.036). A relationship was also found between the score calculated from the parameters' values observed after sperm selection and the fertilization rate (r = 0.21; P = 0.018). In 45 cycles it was possible to assess the acrosomal status of spermatozoa obtained from a 20-JIL sperm suspension from which sperm cells were taken for the injection procedure. Twelve semen samples were exposed for 24 hours to FF, and the mean acrosome reaction rate of the living spermatozoa was 13.9% (range, 4 to 23). In 22 semen samples incubated for 24 hours and exposed to an electric field, the mean value was 18.0% (range, 4 to 44), and in 11 cycles in which the sperm suspension was incubated for up to 4 hours and then processed by electroporation, the percentage of acrosome-free spermatozoa was 12.9% (range, 3 to 55). Only in the protocol in which Vol. 59, No.4, April 1993
electroporation was applied after 24 hours' incubation did the acrosome reaction rate correlate with the fertilization rate after SUZI (r = 0.50; P = 0.018). With the same treatment the score real acrosome reacted (%) X concentration after sperm selection (X106 /mL) X 10- 2 was clearly linked to the fertilization rate (r = 0.64; P = 0.001) obtained after SUZI. Thirty patients became pregnant: 8 pregnancies were preclinical, one clinical abortion occurred at 15 weeks of gestation because of chorion amnionitis, and 1 patient had an ectopic pregnancy requiring a salpingectomy at 7 weeks of gestation. The ongoing and to term PR was 6.7% per started cycle (20/300) and 9.8% per replacement (20/204). There were eight twin gestations (2 of which were vanishing triplets and 1 monovular twin) and 12 singleton pregnancies. In 13 patients, amniocentesis was carried out, and the fetal karyotype of 18 fetuses was normal. Seven patients decided not to have prenatal diagnosis. At the time of writing, 15 patients have delivered 21 babies (6 twin gestations), and 5 pregnancies are ongoing (2 twin). One patient delivered by cesarean section at 38 weeks because of preeclampsia. The child presented dysmaturity and cheilopalatoschisis at birth. From the 21 children born, 4 developed after the replacement of intracytoplasmic sperm injection embryos, 8 after the replacement of SUZI embryos, and 9 after SUZI and intracytoplasmic sperm injection embryos. We examined the ability to implant 294 embryos obtained after assisted fertilization by fertilization with sperm using teratozoospermic samples. Embryos (n = 120) obtained from men with extreme teratozoospermia (::S;4% normal forms) implanted at Palermo et al.
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831
Table 4 Correlation Between Sperm Morphology and Implantation Ability of Embryos Obtained by SUZI and Intracytoplasmic Sperm Injection Percent of normal forms
o to 4
5 to 14
No. of sacs/no. of embryos transferred Technique SUZI* Intracytoplasmic sperm injection * Total
1/43 (2.3)*
lS/134 (13.4)
4/77 (5.2) 5/120 (4.2)t
6/40 (15.0) 24/174 (13.S) t
* Mantel-Haenszel test: difference between the two techniques was not significant; values in parentheses are percents. t Difference between embryo implantation related to sperm morphology, controlled for the technique used, P = 0.007.
a significantly lower rate than embryos (n = 174) from men with less severe teratozoospermia (5% to 14% normal forms). As indicated in Table 4, there was no difference between the SUZI and intracytoplasmic sperm injection procedures. Table 5 summarizes the relationship between sperm characteristics, previous history of assisted reproduction, and fertilization obtained after SUZI and intracytoplasmic sperm injection procedures, including the pregnancies. In all classes of sperm
density, the fertilization rate obtained after the intracytoplasmic sperm injection procedure is clearly better than after the SUZI. Surprisingly, the class 20 to 40 X 106 /mL has a lower fertilization rate using the intracytoplasmic sperm injection procedure than with the others. Twelve pregnancies (only 5 ongoing and to term) appear in the group with sperm density ~ 40 X 106 /mL. Table 6 reports the results of assisted fertilization related to the sperm characteristics in couples without previous IVF because of too poor sperm values. In this series, too, the intracytoplasmic sperm injection-treated oocytes appear to fertilize better than the SUZI-treated, except for the category with the highest density (class> 20 X 106 /mL). The incidence of pregnancies in this group is lower than among the patients with a previous IVF history, whereas the fertilization rate obtained after each of the two injection procedures is similar. DISCUSSION
In this study, 202 couples were treated in 300 consecutive cycles of assisted fertilization. Subzonal insemination was performed in 2,214 oocytes, of which 357 fertilized normally. A new procedure, the intracytoplasmic sperm injection of a single spermatozoon, was performed on 716 oocytes, 240 of which fertilized normally.
Table 5 Fertilization and Pregnancies After Assisted Fertilization Related to Sperm Characteristics in Patients With Previous History of Assisted Reproduction Classes of sperm density*
Sperm characteristics Concentration (X106/mL) Progressive motility (%) Normal forms (%) Outcome of assisted reproduction No. of previous IVF cycles No. of zygotes after IVF/no. of oocytes Outcome of assisted fertilization No. of cycles No. of zygotes/no. of oocytes injected by SUZI No. of zygotes/no. of oocytes after intracytoplasmic sperm injection No. of pregnancies/no. of transfers No. of pregnancies ongoing or to term
,,;1
1 to 5
5 to 20
20 to 40
;0>40
0.5 (0 to 1) t 27.4 (0 to Sl) 3.5 (0 to 12)
3.1 (1.2 to 5) 19.5 (0 to 50) II (0 to 43)
11.7 (5.1 to 20) 26.3 (0 to SO) 14.0 (0 to 47)
30.0 (20.2 to 40) 34.7 (0 to SO) 20.2 (0 to 50)
S1.5 (40.6 to 200) 41.2 (2 to SO) 25.5 (0 to 6S)
14
64
(1 to 3)
6/133 [4.5]:j:
(1 to 6)
35/676 [5.2]
19 7/94
[7.4]
32/74 [43.2] 4/14
Palermo et al.
100/1,032 [9.7]
69
(1 to 6)
32/749 [4.3]
63
llO
(1 to 9)
112/1,223 [9.2] 75
37
30/2S6 [10.5]
64/396
[16.2]
63/296 [21.3]
131/593
[22.1]
26/50 [52.0]
30/63
[47.6]
19/57 [33.3]
42/72
[5S.3]
6/26
3/3S 2
* Density X 106 /mL. t Values are means with ranges in parentheses.
832
(1 to 6)
37
2/21 3
105
Sperm parameters and assisted fertilization
3
12/52 5
5
:j: Values in brackets are percents of fertilization.
Fertility and Sterility
Table 6
Fertilization and Pregnancies After Assisted Fertilization in Patients With Very Poor Semen Characteristics Classes of sperm density'
Sperm characteristics Concentration (X10 6 /mL) Progressive motility (%) Normal forms (%) Epididymal sperm Outcome of assisted fertilization No. of cycles No. of zygotes/no. of oocytes injected by SUZI No. of zygotes/no. of oocytes after intracytoplasmic sperm injection No. of pregnancies/no. of transfers No. of pregnancies ongoing or to term
.,;1
1 to 5
5 to 20
>20
0.3 (0 to l)t 17.0 (0 to 70) 3.5 (0 to 20) One cycle
2.7 (1.1 to 4.9) 15.5 (0 to 63) 4.6 (0 to 24)
8.5 (4.9 to 12.8) 16.8 (0 to 62) 5.9 (0 to 22)
44.8 (34.5 to 58) 2.3 (0 to 6) 23.0 (20 to 28)
29
19
16
4
10/110 [9.1]:j:
23/127 [18.1]
18/77 [23.4]
11/32 [34.4]
55/150 [36.7]
25/56 [44.6]
10/23 [43.5]
1/3 [33.3]
1/22
2/17 1
• Density X 106/mL. t Values are means with ranges in parentheses.
Subzonal insemination can lead to fertilization in previous IVF failures and in cases of extremely impaired semen parameters (8, 15, 22, 23). Furthermore, the SUZI procedure does not result in a significant increase in abnormal embryos (10) or in the birth of abnormal babies (12, 13, 24). In a previous report we have also shown that patients treated by intracytoplasmic sperm injection can achieve pregnancy and give birth to normal babies (14). The main difference between the two techniques is that SUZI requires few but functional spermatozoa, whereas in the intracytoplasmic sperm injection technique, acrosomeless spermatozoa may also be used. Furthermore, in the intracytoplasmic sperm injection technique a single spermatozoon is injected. We observed a better (P = 0.024) fertilization rate after the injection of spermatozoa treated to induce the acrosome reaction than with oocytes injected with untreated spermatozoa (Table 2). Assessment of the acrosomal status of spermatozoa exposed to different treatments showed a mean acrosome reaction rate of 14.9%. In 22 observations in which spermatozoa were treated by an electric field (1,250 Vfcm for 2.5 ms and 24 hours' incubation) and injected subzonally, the oocytes were fertilized in relation to the proportion of acrosome-reacted spermatozoa. In a previous report by our group, a positive correlation was found between the proportion of acrosome-reacted spermatozoa in the sperm suspension and the number of fertilized oocytes obtained after the subzonal insertion of a single mouse spermatozoon (6). Similarly, the cohort of oocytes Vol. 59, No.4, April 1993
0/11
0/3
1 :j: Values in brackets are percents of fertilization.
treated by intracytoplasmic sperm injection showed differences in fertilization. In fact, after one treatment, i.e., electroporation after 24 hours' incubation, the fertilization rate was remarkable. In the oocytes on which intracytoplasmic sperm injection was carried out, untreated spermatozoa fertilized at a rate similar to that of treated spermatozoa. This observation seems to indicate that the treatments applied to induce acrosomal loss affect not only the acrosomal status. Analyzing these data, we were not able to find a clear relationship between a single impaired sperm parameter and the number of oocytes fertilized, the number and quality of the embryos, and the number of pregnancies obtained. By calculating a score taking into account the total number of progressive motile spermatozoa and the frequency of morphologically normal spermatozoa in the ejaculates, we were able to observe a relationship with the number of pregnancies. When the semen score included the sperm values after sperm selection, a correlation with the number of oocytes fertilized by SUZI was found. Furthermore, embryos obtained after the SUZI or intracytoplasmic sperm injection were able to implant at a different rate in relation to the different incidence of morphologically abnormal spermatozoa present in the semen sample. This demonstrates that embryos derived from microsurgical fertilization with extreme teratozoospermia «5% normal forms) were severely impaired (P = 0.0138) in their ability to implant irrespective of whether they were treated by SUZI or intracytoplasmic Palermo et al.
Sperm parameters and assisted fertilization
833
sperm injection. These relative differences in implantation of embryos fertilized with semen with severe teratozoospermia require confirmation on a larger number of pregnancies. These findings are in disagreement with Cohen et al. (15) who claim that the influence of the morphological sperm factor differs in the different microsurgical fertilization procedures used. These assisted fertilization procedures can successfully treat couples who do not conceive after classical IVF or couples with very poor semen parameters. However, some limitations have to be taken into consideration. The enzymatic and mechanical removal of the cumulus-corona cells from the oocytes necessary to allow their manipulation and visualization can be deleterious. This exposure of oocytes to hyaluronidase can induce parthenogenetic activation (23). This phenomenon appears to be more frequent than in the classic IVF (25). The injection procedures per se can damage the oocyte and may also induce parthenogenetic activation. The intracytoplasmic sperm injection procedure can cause more frequent injuries to the oocyte than the SUZI, but the higher fertilization rate after intracytoplasmic sperm injection compensates for this. Although with the intracytoplasmic sperm injection technique we overcome in theory the problem of multiple sperm penetration that besets partial zona dissection and at times SUZI, we sometimes observe the presence of three pronuclei oocytes in the intracytoplasmic sperm injection-treated oocytes; this may be due to nonextrusion of the second polar body. Similarly, embryo cleavage and quality can be influenced by these procedures. This becomes particularly so when very poor semen is used (e.g., in the intracytoplasmic sperm injection) and may also influence the implantation of embryos derived from the injection of poor semen. Although previous reports of our experiences of SUZI and intracytoplasmic sperm injection (13, 14) are encouraging, the evolution of pregnancies obtained by microinjection has to be carefully assessed. At the time of writing, 20 pregnancies have developed to term or are still ongoing. Because few children are born after assisted fertilization, a strict monitoring of the pregnancies by the study of the fetal karyotype and pediatric follow-up ofthe children born is advisable. Acknowledgments. We thank the clinical and scientific staff of the Centre for Reproductive Medicine, Brussels, Belgium, for their expert assistance; Ms. Geertrui Bocken and Ms. An Vankelecom for technical assistance; Ms. Annick Geril and colleagues for counseling; Mr. Frank Winter of the Language Education
834
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Sperm parameters and assisted fertilization
Center for proofreading the text in English; Inge Liebaers, M.D., Ph.D., Department of Medical Genetics of the Dutch-speaking Brussels Free University, Brussels, Belgium, for the genetic counseling and for analyzing the fetal karyotypes; and Ms. Viviane De Wolf for typing the manuscript.
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