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Influence of sperm parameters on embryo quality
FERTILITY AND STERILITY Copyright
©
Vol. 60, No.5, November 1993
Printed on
1993 The American Fertility Society
acid~free
paper in U S. A.
Influence of sperm parameters on embryo quality
Jean Parinaud, M.D., Ph.D.*t:j: Roger Mieusset, M.D., Ph.D.§ Gerard Vieitez*t
Benedicte Labal* Gerard Richoilley, Ph.D.*
Centre Hospitalier Universitaire La Grave, Toulouse, France
Objective: To evaluate the influence of sperm defects on embryo quality. Design: Retrospective study. Setting: In vitro fertilization center. Patients: Embryo transfers (710) from IVF attempts for tubal disease (626) or male infertility (84). Main Outcome Measures: Embryo morphology as a function of causes of infertility, semen, and follicular growth parameters. Embryos were classified into three groups according to their morphology. Results: Transfers of embryos with good morphology were associated to a higher pregnancy rate (34 %) than those with intermediate (24 %) and poor (10%) morphology. Transfers of embryos with a poor morphology were more frequent (26 of 84 versus 114 of 626) and those with a fair aspect were less frequent (24 of 84 versus 229 of 626) in male infertility than in tubal disease. Embryos with a poor morphology were associated with lower percentage of morphologically normal sperms (62% ± 19% versus 67% ± 18%; means ± SD) and a higher percentage of abnormalities of the postacrosomial region (29% ± 15% versus 18% ± 7%). Moreover, sperms with counts < 10 X 106 /mL were associated with a lower percentage of embryos with good morphology (18% versus 37%) than sperms with counts;;>: 10 X 106 /mL. Conclusion: Embryo quality is influenced by the semen quality and especially by sperm head abnormalities, suggesting an important role of the male gamete on the early stages of embryogeneSIS. Fertil Steril 1993;60:888-92 Key words: In vitro fertilization, sperm morphology, embryo morphology
Male factor is well known to induce low pregnancy rate in in vivo (1) and in vitro (2, 3) human reproduction. This reduction in the pregnancy rate is mainly attributed to a low fertilization rate because of either a decreased sperm count and/or motility (2, 3) or a high percentage of spermatozoa with morphological abnormalities (4-6). However,
Received February 8, 1983; revised and accepted July 13, 1993. * Centre de Fecondation In Vitro. t Institut National de la Sante et de la Recherche Medicale, Contrat Jeune Formation 89-08. :j: Reprint requests: Jean Parinaud, M.D., Ph.D., Centre de Fecondation In Vitro, Centre Hospitalier Universitaire, La Grave, 31052 Toulouse Cedex, France. § Centre de Sterilite Masculine.
888
Parinaud et al.
Sperm and embryo quality
impairment of spermatogenesis is not confined to modifications of the usual sperm characteristics. Indeed, the presence of a high number of immature germ cells (7), which are a sensitive indicator ofthe quality ofthe germinative epithelium (8), as well as alterations in DNA of spermatozoa (9) are associated with fertilization failure, despite normal semen parameters. Sperm defects could impair not only fertilization rate but also the fate of embryos, because men from couples with repeated spontaneous abortions have higher sperm chromosome abnormalities than men from fertile couples (10). In vitro fertilization is a good model for observing the influence of the male gamete on embryonic quality because it allows the observation of early embryonic stages and the evaluation of the embryo's ability to develop by studying the pregnancy per Fertility and Sterility
transfer, which rules out fertilization failures. However, data obtained from IVF attempts in cases of male infertility are conflicting, with various reports of no impairment of embryonic development (3), delayed cleavages (11), or poor morphology (12). The present study was designed to evaluate the influence of sperm parameters on embryo quality and pregnancy per transfer rate in IVF. MATERIALS AND METHODS Patients
We studied 710 ETs from patients undergoing an IVF attempt from 1990 to 1992. The causes of infertility were tubal disease (n = 626, 88%) or male infertility (n = 84, 12%). Ovarian Stimulation Procedures
First Step: Pituitary Blockage
Beginning on cycle day 1, patients received subcutaneously LH -RH analogues: buserelin acetate ( [ D - Ser ( But )6 , Pro 9 - NEt] LH - RH , Suprefact; Hoechst, Puteaux, France) 0.3 mg twice per day, triptorelin ([D-Trp6]LH-RH, Decapeptyl; Ipsen, Paris, France) 0.1 mg once per day, or leuprorelin ([D-Leu6,Pro9 -NEt]LH-RH, Lucrin; Abbott, Rungis, France) 0.2 mg once per day. On cycle day 15, plasmatic FSH, LH, and E2 measurements and pelvic ultrasonography were performed to verify pituitary and ovarian blockage. Second Step: Follicular Stimulation
This step began when E2 was <100 pg/mL (conversion factor to SI unit, 3.671). Human menopausal gonadotropins (Humegon; Organon, Paris, France and Neopergonal; Serono, Paris, France) doses varied from 1 to 6 ampules per day. The number of administered ampules was chosen according the endocrine status and the results of previous ovarian stimulations. Follicular growth was assessed daily from stimulation day 10 on by plasmatic E2 measurement and pelvic ultrasonography. Ovulation was induced by injection of 10,000 U hCG 1M when at least three follicles :2: 15 mm and E2 :2: 300 pg/mL (conversion factor to SI unit, 3.671) per growing follicle (:2:15 mm) were obtained. Follicular punctures were ultrasonically guided using an intravaginal probe, 36 hours after hCG injection. Sperm Examination
Sperm parameters were recorded 1 hour after ejaculation. Motility was evaluated by microscopic Vol. 60, No.5, November 1993
examination. After Schorr staining, sperm morphology was classified according to Jouannet et al. (13). Semen Preparation
When semen were normal according to World Health Organization criteria (14), they were prepared using the swim-up method: after two washes in Tyrode's solution (GmCO BRL, Cergy Pontoise, France), sperm were allowed to swim from the pellet up to INRA Menezo B2 medium (Api System, Montalieu Vercieu, France) for 10 minutes at 37°C with 5% CO 2. In other cases, after 30 minutes liquefaction at 37°C, motile spermatozoa were isolated using a discontinuous (60%, 80%, 90%) Percoll (Sigma Chemical Co., St Louis, MO) gradient. After centrifugation for 30 minutes at 200 X g, the 90% Percoll fraction was recovered and sperm cells were then washed once with Tyrode's solution. The final pellet was resuspended in INRA Menezo B2 medium. IVF Procedures
Culture medium was INRA Menezo B2 without any serum or substitute. Oocytes were inseminated with 50,000 spermatozoa. The culture medium was changed after 24 hours and oocytes were then observed for pronuclei assessment. Embryos were transferred 48 hours after follicular puncture (at the two- or four-cell stage) and four embryos were transferred when available. Progesterone (200 mg Utrogestan; Besins-Iscovesco, Paris France) was given intravaginally for 10 days beginning on puncture day. Scoring Embryo Morphology
Embryos were microscopically examined 48 hours after oocyte insemination. The morphologic score was established as follows: score 1, cells with unequal sizes and numerous cytoplasmic fragments; score 2, cells with unequal sizes or presence of few cytoplasmic fragments; score 3, cells with equal sizes and absence of cytoplasmic fragments. Because one to four embryos with different scores were transferred at the same time, the present study used groups of transfers defined as follows: group 1, transfers of only embryos scored 1; group 2, transfers of at least one scored 2 embryo and no scored 3 embryo; group 3, transfers of at least one scored 3 embryo. In our laboratory, these groups were associated with significantly different pregnancy rates. Indeed, when studying 710 transfers Parinaud et al.
Sperm and embryo quality
889
Table 1 Parameters of 1VF Attempts According to the Origin of 1nfertility*
Pregnancies Groups of embryo morphologyt 1 2 3 No. of transfered embryos Age of women (y) Sperm parameters Count (lOB/mL) Motility (%) Normal forms (%) Peak E2 (pg/mL) I No. of follicles ~ 15 mm No. of oocytes Fertilization rate (%)11 Endometrium (mm)**
Characteristics of Patients and Parameters of IVF Attempts According to Embryo Morphology
Tubal disease (n = 626)
Male infertility (n = 84)
156 (25)
16 (19)
114 (18) 283 (43) 229 (37)
26 (31) 34 (41) 24 (29)
1.1:1:
2.9 ± 34.1 ±
4.1
± ± ± ± ± ± ± ±
61 20 17 596 2.4 4.1 26 2
91 56 67 1,708 5.5 7.8 58 12.5
2.3 ± 33.5 ± 44 33 43 1,681 5.9 7.9 43 12.7
± ± ± ± ± ± ± ±
1§ 4 55§ 17§ 19§ 579 2.3 4.3 27§ 1.4
* Values in parenthesis are percents. t Groups of embryo morphology were defined as follows: 1, transfers of only grade 1 embryos; 2, transfers of at least one grade 2 and no grade 3 embryo; 3, transfers of at least one grade 3 embryo. The distribution of groups is significantly different between tubal disease and male infertility (P < 0.05). :I: Values are means ± SD. § Significantly different versus tubal disease (P < 0.001). II Conversion factor to S1 unit, 3.671. 11 Percentage of fertilized oocytes. ** Measured by transvaginal ultrasonography.
from 1990 to 1992, the pregnancy per transfer rate was 21 of 207 (10%) in group 1, 103 of 428 (24%) in group 2, and 115 of219 (34%) in group 3 (P < 0.001). Statistical Comparison
Data are given as means ± SD. Statistical comparisons were made using Student's t-test or x 2 test as appropriate. RESULTS Parameters of IVF Attempts According to the Origin of Infertility
As shown in Table 1, transfers of embryos with a poor morphology were more frequent and those with a fair aspect less frequent in male infertility than in tubal disease (P < 0.05). This was related to a lower, but not significant, pregnancy rate. The difference in embryo morphology seemed not to be due to a lower oocyte quality because female characteristics (age, basal endocrine profile) as well as follicular growth parameters were identical in both populations. 890
Parinaud et al.
Sperm and embryo quality
The quality of embryos had a dramatic influence on pregnancy rates (Table 2), but the number of transferred embryos was lower in group 1 than in groups 2 and 3 (2.1 ± 1.1, 2.9 ± 1.1, and 3.2 ± 0.9, respectively, for groups 1, 2, and 3; P < 0.01). However, with an identical number of transferred embryos the same difference in pregnancy rates was found between the three groups (pregnancy rates when two embryos were transferred: 6 of 63, 19 of 91, and 10 of 30, respectively, for groups 1, 2, and 3; P < 0.05). As reported in Table 2, embryo quality was influenced by both female and male parameters. Despite no difference among groups of embryo quality in maximal plasma E2 and number of mature follicles (~15 mm), the group of poor morphology was associated with a lower number of recovered 00cytes (P < 0.001). Concerning male factors, there was a significant difference in the percentage of normal spermatozoa (P < 0.05) among the groups and a nonsignificant tendency for a higher sperm count in the group of scored 3 embryos. When considering the specific abnormalities of spermatozoa, there was a significant difference for abnormalities of the postacrosomial region (29% ± 15%, 22% ± 13%, and 18% ± 7% for groups 1,2, and 3, respectively; P < 0.05) and a nonsignificant tendency for cytoplasmic droplets (15% ± 10%, 11 % ± 7%, and 10% ± 7% for groups 1, 2, and 3, respectively; P < 0.12); the distribution of the other abnormalities being identical in the three groups. Sperm with counts < 10 X 106 jmL (n = 44; motility = 37% ± 29%; normal forms = 42% ± 20%) were associated with a lower percentage of scored 3 embryos (8 of 44 versus 245 of 666; P < 0.05) than sperm with counts 10 X 106 jmL (n = 666; motility = 55% ± 20%; normal forms = 66% ± 8%). The prevalence of sperm counts < 10 X 106 jmL was 33% in male infertility and 3% in tubal disease (P < 0.001).
DISCUSSION
The reduced fertilization rate reported in the present study in the case of male factor is in agreement with previous studies (2-6). Most authors explained the reduced pregnancy rate observed in male infertility only by the low fertilization rate (2-6). Influence of sperm parameters on embryonic quality has been studied in only one recent report (12), where delayed fertilization was associated Fertility and Sterility
Table 2
Characteristics of Patients and Parameters of IVF Attempts According to Embryo Morphology* Groups of embryo morphologyt
No. of embryo transfers Pregnancies Sperm parameters Count (10"/mL) Motility (%) Normal forms (%) Follicular growth parameters Peak E2 (pg/mL)l1 No. offollic1es
1
2
3
140 13 (9)*
317 78 (25)
253 81 (32)
79 52 62 1,626 5.2 6.9 42 2.1
± 64§ ± 20 ± 1911 ± 588 ± 2.3 ± 4.1** ± 28* ± 1.1*
* Values in parenthesis are percents. t Groups of embryo morphology were defined as follows: ·1, transfers of only grade 1 embryos; 2, transfers of at least one grade 2 and no grade 3 embryo; 3, transfers of at least one grade 3 embryo. P < 0.01 versus groups 2 and 3.
*
with high levels of morphological abnormalities of spermatozoa or with antisperm auto-antibodies. In the present study, the male infertility group was characterized by a significantly lower quality of embryo morphology than in tubal disease. As female characteristics (age, basal endocrine profile, and follicular growth parameters) were similar in both groups, the difference in embryo quality could be related to a male factor rather than to a female one because semen characteristics differ between these two groups. When considering the whole population apart from IVF indications, embryos with a fair morphology were associated with the highest mean percentage of normal spermatozoa, more particularly with normal postacrosomial region. Whatever the IVF indication, impaired spermatogenesis, as shown for example by low sperm count, is associated with a lower number of fair morphology embryos. These data are in good agreement with those of Cohen et al. (6) who reported low pregnancy per transfer rate after micromanipulation techniques in case ofteratozoospermia, suggesting that such abnormal sperm are unable to produce functionally normal embryos. The participation of spermatozoa in the embryo quality was reported in animal experiments with in vitro delayed embryos obtained from heat-stressed spermatozoa (15) and with in vivo increased embryonic loss when ambient (16, 17) or scrotal (18) heating of the male was present. In men, increased levels of chromosome breaks and acentric fragments Vol. 60, No.5, November 1993
83 54 63
± 60 ± 21 ± 2011
1,704 5.6 7.7 56 2.9
± 615 ± 2.6 ± 4.2 ± 24** ± 1.1
91 54 67
± 65 ± 20 ± 18
1,747 ± 567 5.8 ± 2.2 8.4 ± 3.9 66 ± 26 3.2 ± 0.9
§ Values are means ± SD. II P < 0.05 versus group 3. 11 Conversion factor to SI unit, 3.671. ** P < 0.01 versus group 3. tt Percentage of fertilized oocytes.
were reported in sperm of men from couples with unexplained history of repeated spontaneous abortions (10). The embryonic loss could result from a deficiency of genetic material in the embryo secondary to the loss, during the first cleavage, of abnormal male material (10). More recently, denaturation or redistribution and loss of sperm DNA were reported to be present at higher levels in infertile than fertile men (9). Once fertilization is initiated, the sperm DNA must decondense and interact with the oocyte DNA to form the genome of the developing zygote, therefore, embryos resulting from defective sperm DNA could have an abnormal development. However, at the present time, there is no indication that such DNA or chromosome abnormalities in spermatozoa could induce embryos of abnormal quality before the transfer time in IVF, because the control of the embryonic development by the embryo genome begins after the four- to eight-cell stage (19). In conclusion, the results of the present and other (6, 11, 12) studies suggest that the spermatozoa is involved in the embryonic quality, even in the early stages of development, which limits the treatment potency of assisted fertilization procedures in cases of severely impaired spermatogenesis.
REFERENCES 1. Baker HWG, Burger HG, De Kretser D. Male infertility. In: Steinberg E, Frajese G, Steinberger A, editors. Reproductive medicine. New York: Raven Press, 1986:187-97.
Parinaud et al.
Sperm and embryo quality
891
2. Hirsch I, Gibbons WE, Lipshultz LI, Rossavick KK, Young RL, Poindexter AN, et al. In vitro fertilization in couples with male factor infertility. Fertil Steril1986;45:659-64. 3. Van Uem JFHM, Acosta AA, Swanson RJ, Mayer J, Ackerman S, Burkman LJ, et al. Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril1985;44:37583. 4. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehinger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril1988;49:112-7. 5. Simon A, Younis J, Lewin A, Bartoov B, Schenker JG, Laufer N. The correlation between sperm cell morphology and fertilization after zona pellucida slitting in sub fertile men. Fertil Steril1991;56:325-31. 6. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, Reing A, et al. Microsurgical fertilization and teratozoospermia. Hum Reprod 1991;6:118-23. 7. Tomlinson MJ, Barrat CLR, Bolton AE, Lenton EA, Roberts HB, Cooke ID. Round cells and sperm fertilizing capacity: the presence of immature germ cells but not seminal leukocytes are associated with reduced success of in vitro fertilization. Fertil Steril1992;58:1257-9. 8. MacLeod J. Human seminal cytology as a sensitive indicator of the germinal epithelium. Int J Fertil 1964; 9:281-95. 9. Peluso JJ, Luciano AA, Nulsen JC. The relationship between alterations in spermatozoal deoxyribonucleic acid, heparin binding sites and semen quality. Fertil Steril 1992; 57:665-70. 10. Rosenbusch B, Sterzik K. Sperm chromosomes and habitual abortion. Fertil Steril1991;56:370-2. 11. Yovitch JL, Stanger JD. The limitations of in vitro fertiliza-
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Sperm and embryo quality
12.
13.
14.
15.
16.
17.
18.
19.
tion from males with severe oligospermia and abnormal sperm morphology. J In Vitro Fert Embryo Transf 1984; 1:172-9. Ron-El R, Nachum H, Herman A, Golan A, Caspi E, Soffer E. Delayed fertilization and poor embryonic development associated with impaired semen quality. Fertil Steril 1991; 55:338-44. Jouannet P, Ducot B, Feneux D, Spira A. Male factors and likelihood of pregnancy in infertile couples: I study of sperm characteristics. Int J Androl 1988; 11:379-94. World Health Organization. WHO laboratory manual for examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge, United Kingdom: The Press Syndicate of the University of Cambridge, 1987. Burfening PJ, Ulberg LC. Embryonic survival subsequent to culture of rabbit spermatozoa at 38°C and 40°C. J Reprod Fertil 1968; 15:87-92. Bellm'~ AR. Viability and survival of mouse embryos following parental exposure to high temperature. J Reprod Fertil 1972; 30:71-81. Setchell BP, D'Occhio MJ, Hall MJ, Laurie MS, Tucker MJ, Zupp JL. Is embryonic mortality increased in normal females mated to subfertile males? J Reprod Fertil 1988;82:567-74. Mieusset R, Quinta Casares P, Sanchez Partida LG, Sowerbuts SF, Zupp JL, Setchell BP. Effects of heating the testes and epididymides of ram by scrotal insulation on fertility and embryonic mortality in ewes inseminated with frozen sperm. J Reprod FertiI1992;94:337-43. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of pre imp lantation development. Nature 1988; 332:459-61.
Fertility and Sterility
©
Vol. 60, No.5, November 1993
Printed on
1993 The American Fertility Society
acid~free
paper in U S. A.
Influence of sperm parameters on embryo quality
Jean Parinaud, M.D., Ph.D.*t:j: Roger Mieusset, M.D., Ph.D.§ Gerard Vieitez*t
Benedicte Labal* Gerard Richoilley, Ph.D.*
Centre Hospitalier Universitaire La Grave, Toulouse, France
Objective: To evaluate the influence of sperm defects on embryo quality. Design: Retrospective study. Setting: In vitro fertilization center. Patients: Embryo transfers (710) from IVF attempts for tubal disease (626) or male infertility (84). Main Outcome Measures: Embryo morphology as a function of causes of infertility, semen, and follicular growth parameters. Embryos were classified into three groups according to their morphology. Results: Transfers of embryos with good morphology were associated to a higher pregnancy rate (34 %) than those with intermediate (24 %) and poor (10%) morphology. Transfers of embryos with a poor morphology were more frequent (26 of 84 versus 114 of 626) and those with a fair aspect were less frequent (24 of 84 versus 229 of 626) in male infertility than in tubal disease. Embryos with a poor morphology were associated with lower percentage of morphologically normal sperms (62% ± 19% versus 67% ± 18%; means ± SD) and a higher percentage of abnormalities of the postacrosomial region (29% ± 15% versus 18% ± 7%). Moreover, sperms with counts < 10 X 106 /mL were associated with a lower percentage of embryos with good morphology (18% versus 37%) than sperms with counts;;>: 10 X 106 /mL. Conclusion: Embryo quality is influenced by the semen quality and especially by sperm head abnormalities, suggesting an important role of the male gamete on the early stages of embryogeneSIS. Fertil Steril 1993;60:888-92 Key words: In vitro fertilization, sperm morphology, embryo morphology
Male factor is well known to induce low pregnancy rate in in vivo (1) and in vitro (2, 3) human reproduction. This reduction in the pregnancy rate is mainly attributed to a low fertilization rate because of either a decreased sperm count and/or motility (2, 3) or a high percentage of spermatozoa with morphological abnormalities (4-6). However,
Received February 8, 1983; revised and accepted July 13, 1993. * Centre de Fecondation In Vitro. t Institut National de la Sante et de la Recherche Medicale, Contrat Jeune Formation 89-08. :j: Reprint requests: Jean Parinaud, M.D., Ph.D., Centre de Fecondation In Vitro, Centre Hospitalier Universitaire, La Grave, 31052 Toulouse Cedex, France. § Centre de Sterilite Masculine.
888
Parinaud et al.
Sperm and embryo quality
impairment of spermatogenesis is not confined to modifications of the usual sperm characteristics. Indeed, the presence of a high number of immature germ cells (7), which are a sensitive indicator ofthe quality ofthe germinative epithelium (8), as well as alterations in DNA of spermatozoa (9) are associated with fertilization failure, despite normal semen parameters. Sperm defects could impair not only fertilization rate but also the fate of embryos, because men from couples with repeated spontaneous abortions have higher sperm chromosome abnormalities than men from fertile couples (10). In vitro fertilization is a good model for observing the influence of the male gamete on embryonic quality because it allows the observation of early embryonic stages and the evaluation of the embryo's ability to develop by studying the pregnancy per Fertility and Sterility
transfer, which rules out fertilization failures. However, data obtained from IVF attempts in cases of male infertility are conflicting, with various reports of no impairment of embryonic development (3), delayed cleavages (11), or poor morphology (12). The present study was designed to evaluate the influence of sperm parameters on embryo quality and pregnancy per transfer rate in IVF. MATERIALS AND METHODS Patients
We studied 710 ETs from patients undergoing an IVF attempt from 1990 to 1992. The causes of infertility were tubal disease (n = 626, 88%) or male infertility (n = 84, 12%). Ovarian Stimulation Procedures
First Step: Pituitary Blockage
Beginning on cycle day 1, patients received subcutaneously LH -RH analogues: buserelin acetate ( [ D - Ser ( But )6 , Pro 9 - NEt] LH - RH , Suprefact; Hoechst, Puteaux, France) 0.3 mg twice per day, triptorelin ([D-Trp6]LH-RH, Decapeptyl; Ipsen, Paris, France) 0.1 mg once per day, or leuprorelin ([D-Leu6,Pro9 -NEt]LH-RH, Lucrin; Abbott, Rungis, France) 0.2 mg once per day. On cycle day 15, plasmatic FSH, LH, and E2 measurements and pelvic ultrasonography were performed to verify pituitary and ovarian blockage. Second Step: Follicular Stimulation
This step began when E2 was <100 pg/mL (conversion factor to SI unit, 3.671). Human menopausal gonadotropins (Humegon; Organon, Paris, France and Neopergonal; Serono, Paris, France) doses varied from 1 to 6 ampules per day. The number of administered ampules was chosen according the endocrine status and the results of previous ovarian stimulations. Follicular growth was assessed daily from stimulation day 10 on by plasmatic E2 measurement and pelvic ultrasonography. Ovulation was induced by injection of 10,000 U hCG 1M when at least three follicles :2: 15 mm and E2 :2: 300 pg/mL (conversion factor to SI unit, 3.671) per growing follicle (:2:15 mm) were obtained. Follicular punctures were ultrasonically guided using an intravaginal probe, 36 hours after hCG injection. Sperm Examination
Sperm parameters were recorded 1 hour after ejaculation. Motility was evaluated by microscopic Vol. 60, No.5, November 1993
examination. After Schorr staining, sperm morphology was classified according to Jouannet et al. (13). Semen Preparation
When semen were normal according to World Health Organization criteria (14), they were prepared using the swim-up method: after two washes in Tyrode's solution (GmCO BRL, Cergy Pontoise, France), sperm were allowed to swim from the pellet up to INRA Menezo B2 medium (Api System, Montalieu Vercieu, France) for 10 minutes at 37°C with 5% CO 2. In other cases, after 30 minutes liquefaction at 37°C, motile spermatozoa were isolated using a discontinuous (60%, 80%, 90%) Percoll (Sigma Chemical Co., St Louis, MO) gradient. After centrifugation for 30 minutes at 200 X g, the 90% Percoll fraction was recovered and sperm cells were then washed once with Tyrode's solution. The final pellet was resuspended in INRA Menezo B2 medium. IVF Procedures
Culture medium was INRA Menezo B2 without any serum or substitute. Oocytes were inseminated with 50,000 spermatozoa. The culture medium was changed after 24 hours and oocytes were then observed for pronuclei assessment. Embryos were transferred 48 hours after follicular puncture (at the two- or four-cell stage) and four embryos were transferred when available. Progesterone (200 mg Utrogestan; Besins-Iscovesco, Paris France) was given intravaginally for 10 days beginning on puncture day. Scoring Embryo Morphology
Embryos were microscopically examined 48 hours after oocyte insemination. The morphologic score was established as follows: score 1, cells with unequal sizes and numerous cytoplasmic fragments; score 2, cells with unequal sizes or presence of few cytoplasmic fragments; score 3, cells with equal sizes and absence of cytoplasmic fragments. Because one to four embryos with different scores were transferred at the same time, the present study used groups of transfers defined as follows: group 1, transfers of only embryos scored 1; group 2, transfers of at least one scored 2 embryo and no scored 3 embryo; group 3, transfers of at least one scored 3 embryo. In our laboratory, these groups were associated with significantly different pregnancy rates. Indeed, when studying 710 transfers Parinaud et al.
Sperm and embryo quality
889
Table 1 Parameters of 1VF Attempts According to the Origin of 1nfertility*
Pregnancies Groups of embryo morphologyt 1 2 3 No. of transfered embryos Age of women (y) Sperm parameters Count (lOB/mL) Motility (%) Normal forms (%) Peak E2 (pg/mL) I No. of follicles ~ 15 mm No. of oocytes Fertilization rate (%)11 Endometrium (mm)**
Characteristics of Patients and Parameters of IVF Attempts According to Embryo Morphology
Tubal disease (n = 626)
Male infertility (n = 84)
156 (25)
16 (19)
114 (18) 283 (43) 229 (37)
26 (31) 34 (41) 24 (29)
1.1:1:
2.9 ± 34.1 ±
4.1
± ± ± ± ± ± ± ±
61 20 17 596 2.4 4.1 26 2
91 56 67 1,708 5.5 7.8 58 12.5
2.3 ± 33.5 ± 44 33 43 1,681 5.9 7.9 43 12.7
± ± ± ± ± ± ± ±
1§ 4 55§ 17§ 19§ 579 2.3 4.3 27§ 1.4
* Values in parenthesis are percents. t Groups of embryo morphology were defined as follows: 1, transfers of only grade 1 embryos; 2, transfers of at least one grade 2 and no grade 3 embryo; 3, transfers of at least one grade 3 embryo. The distribution of groups is significantly different between tubal disease and male infertility (P < 0.05). :I: Values are means ± SD. § Significantly different versus tubal disease (P < 0.001). II Conversion factor to S1 unit, 3.671. 11 Percentage of fertilized oocytes. ** Measured by transvaginal ultrasonography.
from 1990 to 1992, the pregnancy per transfer rate was 21 of 207 (10%) in group 1, 103 of 428 (24%) in group 2, and 115 of219 (34%) in group 3 (P < 0.001). Statistical Comparison
Data are given as means ± SD. Statistical comparisons were made using Student's t-test or x 2 test as appropriate. RESULTS Parameters of IVF Attempts According to the Origin of Infertility
As shown in Table 1, transfers of embryos with a poor morphology were more frequent and those with a fair aspect less frequent in male infertility than in tubal disease (P < 0.05). This was related to a lower, but not significant, pregnancy rate. The difference in embryo morphology seemed not to be due to a lower oocyte quality because female characteristics (age, basal endocrine profile) as well as follicular growth parameters were identical in both populations. 890
Parinaud et al.
Sperm and embryo quality
The quality of embryos had a dramatic influence on pregnancy rates (Table 2), but the number of transferred embryos was lower in group 1 than in groups 2 and 3 (2.1 ± 1.1, 2.9 ± 1.1, and 3.2 ± 0.9, respectively, for groups 1, 2, and 3; P < 0.01). However, with an identical number of transferred embryos the same difference in pregnancy rates was found between the three groups (pregnancy rates when two embryos were transferred: 6 of 63, 19 of 91, and 10 of 30, respectively, for groups 1, 2, and 3; P < 0.05). As reported in Table 2, embryo quality was influenced by both female and male parameters. Despite no difference among groups of embryo quality in maximal plasma E2 and number of mature follicles (~15 mm), the group of poor morphology was associated with a lower number of recovered 00cytes (P < 0.001). Concerning male factors, there was a significant difference in the percentage of normal spermatozoa (P < 0.05) among the groups and a nonsignificant tendency for a higher sperm count in the group of scored 3 embryos. When considering the specific abnormalities of spermatozoa, there was a significant difference for abnormalities of the postacrosomial region (29% ± 15%, 22% ± 13%, and 18% ± 7% for groups 1,2, and 3, respectively; P < 0.05) and a nonsignificant tendency for cytoplasmic droplets (15% ± 10%, 11 % ± 7%, and 10% ± 7% for groups 1, 2, and 3, respectively; P < 0.12); the distribution of the other abnormalities being identical in the three groups. Sperm with counts < 10 X 106 jmL (n = 44; motility = 37% ± 29%; normal forms = 42% ± 20%) were associated with a lower percentage of scored 3 embryos (8 of 44 versus 245 of 666; P < 0.05) than sperm with counts 10 X 106 jmL (n = 666; motility = 55% ± 20%; normal forms = 66% ± 8%). The prevalence of sperm counts < 10 X 106 jmL was 33% in male infertility and 3% in tubal disease (P < 0.001).
DISCUSSION
The reduced fertilization rate reported in the present study in the case of male factor is in agreement with previous studies (2-6). Most authors explained the reduced pregnancy rate observed in male infertility only by the low fertilization rate (2-6). Influence of sperm parameters on embryonic quality has been studied in only one recent report (12), where delayed fertilization was associated Fertility and Sterility
Table 2
Characteristics of Patients and Parameters of IVF Attempts According to Embryo Morphology* Groups of embryo morphologyt
No. of embryo transfers Pregnancies Sperm parameters Count (10"/mL) Motility (%) Normal forms (%) Follicular growth parameters Peak E2 (pg/mL)l1 No. offollic1es
1
2
3
140 13 (9)*
317 78 (25)
253 81 (32)
79 52 62 1,626 5.2 6.9 42 2.1
± 64§ ± 20 ± 1911 ± 588 ± 2.3 ± 4.1** ± 28* ± 1.1*
* Values in parenthesis are percents. t Groups of embryo morphology were defined as follows: ·1, transfers of only grade 1 embryos; 2, transfers of at least one grade 2 and no grade 3 embryo; 3, transfers of at least one grade 3 embryo. P < 0.01 versus groups 2 and 3.
*
with high levels of morphological abnormalities of spermatozoa or with antisperm auto-antibodies. In the present study, the male infertility group was characterized by a significantly lower quality of embryo morphology than in tubal disease. As female characteristics (age, basal endocrine profile, and follicular growth parameters) were similar in both groups, the difference in embryo quality could be related to a male factor rather than to a female one because semen characteristics differ between these two groups. When considering the whole population apart from IVF indications, embryos with a fair morphology were associated with the highest mean percentage of normal spermatozoa, more particularly with normal postacrosomial region. Whatever the IVF indication, impaired spermatogenesis, as shown for example by low sperm count, is associated with a lower number of fair morphology embryos. These data are in good agreement with those of Cohen et al. (6) who reported low pregnancy per transfer rate after micromanipulation techniques in case ofteratozoospermia, suggesting that such abnormal sperm are unable to produce functionally normal embryos. The participation of spermatozoa in the embryo quality was reported in animal experiments with in vitro delayed embryos obtained from heat-stressed spermatozoa (15) and with in vivo increased embryonic loss when ambient (16, 17) or scrotal (18) heating of the male was present. In men, increased levels of chromosome breaks and acentric fragments Vol. 60, No.5, November 1993
83 54 63
± 60 ± 21 ± 2011
1,704 5.6 7.7 56 2.9
± 615 ± 2.6 ± 4.2 ± 24** ± 1.1
91 54 67
± 65 ± 20 ± 18
1,747 ± 567 5.8 ± 2.2 8.4 ± 3.9 66 ± 26 3.2 ± 0.9
§ Values are means ± SD. II P < 0.05 versus group 3. 11 Conversion factor to SI unit, 3.671. ** P < 0.01 versus group 3. tt Percentage of fertilized oocytes.
were reported in sperm of men from couples with unexplained history of repeated spontaneous abortions (10). The embryonic loss could result from a deficiency of genetic material in the embryo secondary to the loss, during the first cleavage, of abnormal male material (10). More recently, denaturation or redistribution and loss of sperm DNA were reported to be present at higher levels in infertile than fertile men (9). Once fertilization is initiated, the sperm DNA must decondense and interact with the oocyte DNA to form the genome of the developing zygote, therefore, embryos resulting from defective sperm DNA could have an abnormal development. However, at the present time, there is no indication that such DNA or chromosome abnormalities in spermatozoa could induce embryos of abnormal quality before the transfer time in IVF, because the control of the embryonic development by the embryo genome begins after the four- to eight-cell stage (19). In conclusion, the results of the present and other (6, 11, 12) studies suggest that the spermatozoa is involved in the embryonic quality, even in the early stages of development, which limits the treatment potency of assisted fertilization procedures in cases of severely impaired spermatogenesis.
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