Assisted reproductivetechnology

FEHTILITY A."lD STERILIrr®

Copyright " 1997 American Society for Reproductive Medicine

Published by Elsevier Science Inc.

Vol. 68, No. 1, July 1997

Printed on acid -free paper in U. S. A.

Fertilization and pregnancy rates after intracytoplasmic sperminjection using ejaculate semen and surgically retrieved sperm

Mohamed A. Aboulghar, M.D.*t:l:Ragaa T. Mansour, M.D.*Gamal 1. Serour, M.D.*Ibrahim Fahmy, M.D.t

Ahmed Kamal , M.D.*Nevine A. Tawab, B.Sc.*Yahia M. Amin, M.D.*

The Egyptian NF-ET Center, Maadi, Cairo, Egypt

Objective: To compare the fertilization rates and pregnancy rates (PRs) in intracytoplasmicsperm injection (ICSI) using sperm from ejaculates of normal and abnormal semen, epididymalsperm , and testicular sperm of obstructive and nonobstructive azoospermic patients.

Design: Retrospective study.Setting: The Egyptian IVF-ET Center.Patientts): Three hundred fifty patients underwent 366 ICSI cycles.Intervention(s): ICSI , epididymal sperm aspiration, and testicular biopsy.Main Outcome Measure(s): Fertilization rates and PRs .Result(s): Patients were divided into five groups according to the quality and source of sperm.

Patients in group 1 underwent 102 cycles of IeSI using ejaculated abnormal semen , group 2underwent 44 cycles using epididymal sperm, group 3 underwent 82 cycles using testicularsperm from obstructive azoospermia, group 4 underwent 80 cycles using testi cular sperm fromnonobstructive azoospermia, and group 5 underwent 58 cycles using normal semen. There wasno significant difference in the fertilization rates and PRs among groups 1, 2, and 3. In group4, the fertilization rate and PR were significantly lower than in all other groups. In group 5,the fertilization rate was significantly high er than in all other groups.

Conclusionfs): The fertilizing ability of sperm in ICSI is highest with normal semen andlowest with sperm extracted from a testicular biopsy in nonobstructive azoospermia. There wasno significant difference in fertilization rates and PRs between ejaculated sperm of differentparameters and surgically retrieved sperm in obstructive azoospermia. (Fertil Steril®1997;68:108-11. © 1997 by American Society for Reproductive Medicine.)

Key Words: ICSI , obstructive azoospermia, nonobstructive azoospermia, MESA, TESE, tes­ticular sperm , epididymal sperm

The first successful pregnancies after intracy­toplasmic sperm injection (IGS!) in humans werereported by Palermo et al. (1). In a large series, VanSteirteghem et al. (2) reported a high success rateusing IGSI in male factor infertility. After publica­tion of this report, IGSI became a standard treat­ment of male factor infertility irrespective of the se-

Received October 31, 1996; revised and accepte d March 5, 1997.* The Egyptian NF-ET Center .t Cairo University.t Reprint requests: Mohamed Aboulghar, M.D., The Egyptian

IVF-ET Center, 85, Maadi Zeraie Road , Maadi, Cairo 11431,Egypt (FAX: 202-351-6649),

108

verity of the condition (3). The scope ofIGSI widenedto include patients with acrosomeless spermatozoa(4), unexplained infertility (5), borderline semen (6),immunologic infertility (7), and previous failure offertilization in conventional IVF (8).

Patients with obstructive (9) and nonobstructive(10, 11) azoospermia also were treated successfullywith IGSI using surgically retrieved sperm from theepididymis or testis.

Different sperm sources having different spermparameters representing various etiologic conditionswere used for IGSI procedures. The aim ofthe pre s­ent work is to compare the fertilization rates, preg­nancy rates (PRs), and outcome in IGSI using sperm

0015-0282/97 /$17.00PI! 80015-0282(97)00070-8

Table 1 Age of the Female Partner and Duration of Infertilityin All Groups

No. of Period ofGroup patients Age* infertility*

y y

1t 102 35.1 ± 3.9 8.1 ± 3.82+ 37 33.6 ± 4.8 9.5 ± 5.9-r-

3§ 74 33.7 ± 4.7 9.8 ± 5.8411 79 33.8 ± 4.7 9.6 ± 5.85~ 58 35.5 ± 4.8 7.7 :!: 4.3

* Values are means ± SD.t Group 1, ejaculated sperm.t Group 2, epididymal sperm (obstructive azoospermia).§ Group 3, testicular sperm (obstructive azoospermia).1\ Group 4, testicular sperm (nonobstructive azoospermia).~ Group 5, normal semen.

of ejaculated normal and abnormal semen, epididy­mal sperm, and testicular sperm of obstructive andnonobstructive azoospermia.

MATERIALS AND METHODS

This is a retrospective study of 350 patients whounderwent 366 cycles of ICSI over a period of 18months. Our standard IVF and ICSI program wasthe same during this period. According to the indica­tions of ICSI, patients were divided into five groupsaccording to the quality and source of sperm. Pa­tients in group 1 had male factor infertility and un­derwent 102 ICSI cycles using ejaculated sperm. Pa­tients in group 2 had obstructive azoospermia andunderwent 44 cycles using sperm retrieved from theepididymis. Patients in group 3 had obstructiveazoospermia and underwent 82 cycles using spermretrieved from testicular biopsy, including testicularcryopreserved sperm used in 6 cycles. Patients ingroup 4 had nonobstructive azoospermia and under­went 80 cycles in which testicular biopsy was donein an attempt to retrieve testicular sperm. Patientsin group 5 had tubal factor infertility with normalsemen parameters and underwent 58 cycles so thatwe could evaluate the possible value of ICSI in thesepatients (12). The patients' characteristics areshown in Table 1. Groups 2, 3, 4, and 5 included allcycles performed in our center during the period ofthe study. In group 1, only the first 102 consecutivecycles were included.

Ovarian Stimulation and Oocyte Retrieval

The female partners received 200 j.lg/8 h buserelinacetate nasal spray (Suprefact; Hoechst AG, Frank­furt, Germany) 10 days before menstruation and untilthe day ofhCG injection. Serum E2level was measured2 weeks after the start of GnRH agonist (GnRH-a). Ifthe E2 level was ::::;40 pg/mL (conversion factor to SI

Vol. 68, No.1, July 1997

unit, 3.671),hMG was started; otherwise, GnRH-a wascontinued for 1 more week, during which the E2 levelwas remeasured before starting hMG. Human meno­pausal gonadotropin (Humegon; Organon, Oss, TheNetherlands) was given 150 IU/d 1M for 5 days andthen the dose was modified according to the response.When two or more follicles reached 2:18 mm in meandiameter, 10,000 ill hCG was given 1Mand ovum pick­up was scheduled 36 hours later. Our techniques ofsemen preparation (13), microsurgical epididymalsperm aspiration and testicular biopsy processing (11),and oocytehandling and micromanipulation (14) werepublished previously.

Two weeks after ET, serum ,B-hCG was measured,and clinical pregnancy was diagnosed by the presenceof fetal sac, echoes, and pulsations 3 weeks later.

Statistical Analysis

Comparisons between groups with respect to per­centage offertilized oocytes, cycles reaching ET, andPR per puncture and per ET were done using the X2

test. P values < 0.05 were considered significant.

RESULTS

There was no statistically significant differencebetween the woman's age or duration of infertilityin all groups (Table 1).

Sperm Parameters According to Sources ofRetrieval of Sperm

Group 1 included 49 patients with a count < 1X 106 mL, 32 patients who had a count from 1 to 10X 106/mL, and 21 patients with a count from 10 to20 X 106/mL. In group 2, the epididymal samplesaspirated contained a large number of spermatozoain most of cases, ranging between 0.3 and 200 X 106

/

mL, with an average of 30 X 106/mL. The initialsperm motility ranged between 1% and 25%, withan average of 6% and forward grade 1 and 2 ac­cording to World Health Organization standards(15), The abnormal forms ranged from 65% to 100%according to strict criteria (16).

In groups 3 and 4, testicular sperm ranged fromvery few spermatozoa to 2 x 106/mL, with an averageof 5% motility and forward grade 1+ or barelytwitching in place. In 29 cycles of nonobstructiveazoospermia, even after an extensive search up to 4hours, no spermatozoa were found.

In group 5, all semen samples were normal andthe cause of infertility was diagnosed as tubal factor.

Table 2 shows the number of oocytes injected, thefertilization rate, and the PR and outcome in allgroups.

There was no significant difference in the fertiliza-

Aboulghar et al, ICSI with sperm of different sources 109

Table 2 Fertilization Rates, PRs, and Outcome in all Groups

Group 1 Group 2 Group 3 Group 4 Group 5

No. of cycles 102 44 82 80 58No. of injected MIl oocytes 846 472 824 489* 572No. of fertilized oocytest 482 (56.9) 260 (55.1) 432 (52.4) 184 (37.6Jt 400 (70)+No. of cycles reaching ETt 102 (99) 44 (100) 82 (100) 51 (63)+ 58 (100)Average no. of embryos per transfer 3.5 3.7 3.6 3.6 3.9No. of pregnancies§ 35 (34.3) 16 (36.4) 28 (34.1) 11 (13.7)+ 19 (32.8)PR per ET (%) 34.7 36.4 34.2 21.6 32.8No. of multiple pregnancies'[ 9 (25.7) 5 (31.3) 6 (21.4) 2 (18.2) 5 (26.3)No. of abortions] 7 (20) 3 (18.8) 4 (14.3) 0 4 (21)

P Value

<0.001<0.001

0.0013

*Three hundred twenty-three oocytes from 29 cycles were notinjected because of failure to retrieve sperm.

t Values in parentheses are percentages.

tion rates and PRs per puncture between groups 1,2, and 3. In nonobstructive azoospermia (group 4),the fertilization rate and PR per puncture were sig­nificantly lower than in all groups (P < 0.001 and P< 0.013, respectively).

In patients with normal semen (group 5), the fer­tilization rate was significantly higher than in allother groups (P < 0.001), but there was no signifi­cant difference in PR per puncture between group 5and groups 1, 2, and 3. There was no significantdifference in the PR per ET in all groups.

In group 1, the fertilization rates and PRs werenot affected by different semen parameters (13). Themultiple PR and the abortion rate between allgroups could not be evaluated statistically becauseof the small numbers.

DISCUSSION

In conventional IVF, it was reported that embryosresulting from poor semen had lower quality andwere associated with a low PR (17). Before the intro­duction of !CSI, it had been shown that sperm char­acteristics influenced the outcome of assisted fertil­ization techniques and that mostly teratozoospermicsemen appeared to be unable to produce functionallynormal embryos, either because of metabolic errorsor because of genetic aberrations (18). Using ejacu­lated sperm for ICSI in the treatment of male factorinfertility showed that there was no correlation be­tween sperm motility, density, or percentage of ab­normal forms and the fertilization rates and PRs aslong as motile, morphologically well shaped spermcould be found for the microinjection (13).

The present study demonstrated that there wasno significant difference in the fertilization rate orPR between ICSI using ejaculated sperms or surgi­cally retrieved sperms in cases of obstructiveazoospermia. Also, there was no significant differ­ence in the fertilization rate and PR achieved byusing epididymal or testicular sperm. These results

110 Aboulghar et aI. 1GS1 with sperm of different sources

:j: Group significantly different from other groups.§ Values in parentheses are PRs per puncture, expressed as

percentages.

showed that the epididymis had no role in the fertil­izing ability ofthe sperm in ICSI. However, this doesnot apply to its possible role in fertilization in vivo.

In our initial experience, we used microsurgicalepididymal sperm aspiration (MESA) for all cases ofobstructive azoospermia, and if epididymal aspira­tion failed or was impossible, testicular biopsy wasperformed. After realizing that testicular sperm pro­duced results that were comparable to those of epi­didymal sperm, we changed to testicular sperm ex­traction (TESE) in all cases. The procedure ofTESEis relatively simple, can be done under local anesthe­sia, and requires less training, shorter operativetime, and no special equipment. However, Silber etaL (19) reported that the number and motility oftesticular sperm may be too low to allow additionalbenefit offreezing. In the present study we were ableto cryopreserve the rest of the testicular sperm. Insix cycles, cryo-thawed testicular spermatozoa wereused, and all of these spermatozoa reached the ETstage, with the achievement of two pregnancies.When a spermatocele was present or when the epi­didymis was found distended, in cases of congenitalabsence of the vas, we resorted to percutaneous fine­needle aspiration to obtain spermatozoa (20).

Our data have shown that in nonobstructiveazoospermia, fertilization rate was 22.6% per re­trieved oocyte and 37.6% per injected oocyte and PRper puncture was 13.7%, compared with fertilizationrate of 52.4% and PR of 34.1% in obstructiveazoospermia. The difference was statistically signifi­cant. However, there was no statistically significantdifference between PR per ET in the two groups. Inthe nonobstructive group, only 63.8% reached the ETstage, compared with 100% in the obstructive group.

Patients diagnosed with nonobstructive azoosper­mia because of Sertoli-cell-only syndrome or matu­ration arrest have occasional foci of normal seminif­erous tubules, with spermatogenesis in more thanhalf of the cases (21).

Fertility and Sterility»

In our study, in 63.8% of patients with nonobstruc­tive azoospermia, sperm could be retrieved from thetesticular biopsy after a search of up to 4 hours insome cases.

Even in nonobstructive azoospermia with elevatedserum FSH level, pregnancy was reported recently(22). Therefore, lCSl using testicular biopsy in non­obstructive azoospermia should not be denied onlyon the basis of elevated FSH or previous biopsy re­port. However, proper counseling about the possibil­ity of finding sperm and about the fertilization rateand PR in these patients, taking into considerationthe age of the female partner, is essential beforeattempting the procedure.

There is a general consensus that infants bornafter lCSl should be karyotyped and followed up todetect any possible increase in incidence of abnor­malities (23).

The fertilization rate in patients with normal se­men (group 5) was significantly higher than in allother groups; however, the PR was not different fromthat in groups 1, 2, and 3. This may suggest thatfailure of fertilization in a percentage of the injectedoocytes in lCSI might be because of defects in thesperm even if it appeared morphologically normal.The present results seem to show that the fertilizingability of sperm in lCSl is highest with normal se­men and lowest with sperm retrieved from nonob­structive azoospermia. However, lCSl did not proveto be superior to conventional lVF in patients withtubal factor infertility with normal semen (12).

In conclusion, our study demonstrated that in us­ing ICSI for the treatment of male factor infertility,there was no significant difference in the fertiliza­tion rates or PRs between different parameters ofsperm in the ejaculate and sperm retrieved from tes­tis or epididymis in obstructive azoospermia. Usingnormal semen for lCSl, the fertilization rate wassignificantly higher than in all other groups. Fertil­ization rates and PRs were lowest in patients withnonobstructive azoospermia.

REFERENCES

1. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Preg­nancies after intracytoplasmic injection of single spermato­zoon into an oocyte. Lancet 1992;340:17 -8.

2. Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, SmitzJ, et al. High fertilization and implantation rates after intracy­toplasmic sperm injection. Hum Reprod 1993;8:1061-6.

3. Mansour R. Gamete micromanipulation and assisted fertil­ization. Middle East Fertil Soc J 1996; 1:91-100.

4. Lundin K, Sjorgren A, Nilsson L, Hamberger L. Fertilizationand pregnancy after intracytoplasmic microinjection of aero­someless spermatozoa. Fertil Steril1994;62:1266-7.

5. Aboulghar MA, Mansour RT, Serour GI, Sattar MA, Amin Y.Intracytoplasmic sperm injection and conventional in vitro fer­tilization for sibling oocytes in cases of unexplained infertilityand borderline semen. J Ass Reprod Genet 1995;13:38-42.

6. Aboulghar MA, Mansour RT, Serour GI, Amin Y. The role ofintracytoplasmic sperm injection (ICSIl in the treatment of pa­tients with borderline semen. Hum Reprod 1995;10:2829-30.

7. Nagy ZP, Verheyen G, Liu J, Joris H, Janssenswillen C, Wi­santo A, et al. Results of 55 intracytoplasmic sperm injectioncycles in the treatment of male-immunological infertility.Hum Reprod 1995; 10:1775-80.

8. Cohen J, Alikani M, Munne S, Palermo GD. Micromanipula­tion in clinical management of fertility disorders. Semin Re­prod Endocrinol1994; 12:151-68.

9. Tournaye H, Devroey P, Liu J, Nagy Z, Lissens W, Van Steir­teghem AV. Microsurgical epididymal sperm aspiration andintracytoplasmic sperm injection: a new effective approachto infertility as a result of congenital bilateral absence of thevas deference. Fertil Steril 1994;61:1045-51.

10. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, TournayeH, Devroey P. High fertilization and pregnancy rate afterintracytoplasmic sperm injection with spermatozoa obtainedfrom testicle biopsy. Hum Reprod 1995;10:148-52.

11. Mansour RT, Aboulghar MA, Serour GI, Fahmi I, Ramzi AM,Amin Y. Intracytoplasmic sperm injection using microsurgi­cally retrieved epididymal and testicular sperm. Fertil Steril1996;65:566-72.

12. Aboulghar MA, Mansour RT, Serour GI, Amin Y, Kamal A.Prospective controlled randomized study of in vitro fertiliza­tion versus intracytoplasmic sperm injection in the treatmentof tubal factor infertility with normal semen parameters. Fer­til Steril 1996;66:753-6.

13. Mansour RT, Aboulghar MA, Serour GI, Amin Y, Ramzi AM.The effect of sperm parameters on the outcome of intracy­toplasmic sperm injection. Fertil Steril 1995;64:982-6.

14. Mansour RT, Aboulghar MA, Serour GI, Tawab N, Amin Y,Sattar M. Successful intracytoplasmic sperm injection with­out performing cytoplasmic aspiration. Fertil Steril 1996;66:256-9.

15. World Health Organization. Laboratory manual for the ex­amination of human semen and sperm-cervical mucus inter­action. 3rd ed. New York: Cambridge University Press,1993;13-80.

16. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF,Oehninger S. Predictive value of abnormal sperm morphologyin in vitro fertilization. Fertil Steril 1998;49:112-7.

17. Parinaud J, Mieusset R, Vietez G, Labal B, Richoilley G.Influence of sperm parameters on embryo quality. FertilSteril 1993;60:888-92.

18. Cohen J, Talansky BE, Malter H, Alikani M, Adler A, ReingA, et al. Microsurgical fertilization and teratozoospermia.Hum Reprod 1991;6:118-23.

19. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, TournayeH, Devroey P. High fertilization and pregnancy rate afterintracytoplasmic sperm injection with spermatozoa obtainedfrom testicle biopsy. Hum Reprod 1995;10:148-52.

20. Craft I, Tsirigotis M, Bennet V, Taranissi M, Khalifa Y, Ho­gewind G, et al. Percutaneous epididymal sperm aspirationand intracytoplasmic sperm injection in the management ofinfertility due to obstructive azoospermia. Fertil Steril 1995;63:1038-42.

21. Silber SJ, Van Steirteghem A, Devroey P. Sertoli cell onlyrevisited. Hum Reprod 1995;10:1031-2.

22. Gil-Salom M, Remohi J, Minguez Y, Rubio C, Pellicer A. Preg­nancy in an azoospermic patient with markedly elevated se­rum follicle-stimulating hormone levels. Fertil Steril 1995;64:1218-20.

23. Bonduelle M, Legein J, Buysse A, Van Assche E, Wisanto A,Devroey P, et al. Prospective follow-up study of 432 childrenborn after intracytoplasmic sperm injection. Hum Reprod1996;11:101-7.

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