Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection*

Transcription

1 FERTILITY AND STERILITY Copyright 1996 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection* Roger Abdelmassih, M.D. Soraya Sollia, M.L.T. Marcelo Moretto, M.D. Anibal A. Acosta, M.D.t Clfnica e Centro de Pesquisa em Reprodur;ilo Humana "Roger Abdelmassih," Silo Paulo, Brazil Objective: To assess the results obtained in our clinic with intracytoplasmic sperm injection (ICSI) in severe male factor infertility and failed fertilization and to determine ifthe age of the female has any impact on those results. Design: Retrospective study. Setting: Private high-complexity Human Reproduction Center. Patients: One hundred five couples with a total of 114 procedures. Eighty-six were classified as severe male factor and 19 were classified as previous failed fertilization. Interventions: Treatment was performed by ICSI. Main Outcome Measure: Normal fertilization, cleavage, and implantation, total, term, and ongoing pregnancy rates in the total population and in different age brackets. Results: Excellent fertilization, cleavage, and implantation rates were obtained with this procedure (78%, 85%, and 13.5%, respectively). The total pregnancy rates were 43% and 46% per cycle and per transfer and 31.5% and 33.6% in terms of term and ongoing pregnancy rates. A significant reduction in implantation and total, and term and ongoing pregnancy rates was seen after the age of 35 years. Conclusions: The results obtained with ICSI are quite satisfactory with proper equipment and careful training. The age of the female is an important parameter in determining prognosis and treatment outcome. Fertil Steril 1996;65:573-7 Key Words: Microinjection, reproduction techniques, fertilization in vitro, maternal age Because of the reintroduction of intracytoplasmic sperm injection (IeSl) in the literature by the group in Brussels (1), a great interest in the technique has developed and many centers are using the procedure mainly for either severe male factor infertility that prevents fertilization if conventional (standard) IVF is used or previous failed fertilization in standard IVF in a laboratory obtaining fertilization rates in excess of 60%. Received May 30, 1995; revised and accepted September 13, * Presented at the 9th World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, April 3 to 7, t Reprint requests: Anibal A. Acosta, M.D., Rua Maestro Elias Lobo, , Sao Paulo, SP, Brazil (FAX: ). This method has shown a high degree of success in large centers but it remains to be demonstrated whether these results can be reproduced in smaller units with fewer cases available. The quality of the sperm used does not seem to influence the outcome (2), but other factors that can help in predicting results have not been established clearly. Furthermore, the sperm parameters and tests of sperm function that can be used as guidelines to indicate whether conventional IVF or IeSI is appropriate are not described clearly. The indications have for the most part been restricted to the situations mentioned above, but there are some groups supporting the notion of broadening the list of conditions in which the procedure can be indicated. (Lisi F, Fishel S, Rinaldi L, Lisi R, Rinaldi A, abstract). We have reviewed the experience of our clinic with the use Abdelmassih et al. Female age and ICSI 573

2 of this technique and report that female age is a significant prognostic factor in predicting outcome. Patient Population MATERIALS AND METHODS After adequate equipment and proper training of the laboratory personnel was obtained, our group started the clinical work in May This review includes the cases performed through January 1995 in the two main indications previously mentioned: a severe male factor «5 X 10 6 spermlml and/or <20% progressive motility and/or <10% normal morphology using World Health Organization criteria [3] in the original specimen) or previous failed fertilization in our clinic or other centers. One hundred five couples had a total of 114 ICSI procedures. Eighty-six couples represented severe male factors and 19 had failed fertilization in a previous IVF attempt (8 in our clinic and 11 in other centers). The female patients mean age was 35.2 years. Ovarian Stimulation Ovarian stimulation was performed using a GnRH agonist long protocol (leuprolide acetate, LA, Lupron; Abbott Laborat6rios do Brasil Ltd, Sao Paulo, Brazil), hmg (Pergonal; Serono Laboratorios, Mexico DF, Mexico), pure FSH (Metrodin; Serono Laboratorios), or a combination of the two. Monitoring was performed using daily serum E2 determinations by time-resolved fluoroimmunoassay (Delfia; Wallac Oy, Turku, Finland) and transvaginal ultrasound (VS) evaluation of follicular development using the average of the two largest diameters as routine measurements. Human chorionic gonadotropin (10,000 IV Profasi; Serono Laboratorios) was administered when at least two 18-mm follicles were visualized. Transvaginal ultrasonically guided oocyte retrieval was performed 34 hours thereafter. Embryology Laboratory Procedure The oocytes, collected in pure follicular fluid, were sent immediately to the adjacent Embryology Laboratory where they were identified using a dissecting microscope at X 50 magnification; they were put in a single-well culture dish (3260; Costar, Cambridge, MA) in human tubal fluid (HTF) culture medium equilibrated overnight and supplemented with 15% patient's preovulatory serum, covered with light mineral oil (Fisher Scientific, Fair Lawn, NJ), and incubated for 4 hours at 37 C in 5% CO2 in air atmosphere. At the end of the incubation period, the cu- mulus and corona radiata cells were removed by incubation in modified HTF with HEPES buffer with 80 IVlmL hyaluronidase type VIII (Sigma Chemical Co., St. Louis, MO) for 30 seconds. They then were washed several times in HTF with 15% preovulatory patient serum. Diagnosis of oocyte stage of development and quality was performed by evaluation of polar body, nuclear status, and morphological characteristics ofthe oocyte and zona pellucida using an inverted microscope (Nikon Diaphot Microscope; Nikon Corporation, Tokyo, Japan) at x200 to 400 magnification. Metaphase II (MIl) oocytes were microinjected within a period of 4 hours of collection; metaphase I (M!) oocytes were observed for the extrusion of the first polar body at 4-hour intervals up to 8 hours after retrieval and the injection was performed accordingly. If a polar body was not extruded up to that time the microinjection procedure was done in the next morning. Microinjection is performed following the techniques and recommendations of the Brussels group (4) using manufactured holding (k HPIP-1000) and injection pipets (k-mpip-1035) (WA Cook Australia PTY Ltd, Eight Mile Plains, Queensland, Australia). The external and internal diameters were 60 to 80, 10, 7 to 8, and 5 to 6 flm for the holding and injection pipets, respectively. Both are angled at 45 to facilitate injection. Sperm is collected at the time of the procedure. Information about sperm quality is known already through a previous complete basic semen analysis. A quick second basic semen analysis is performed in the embryology laboratory after liquefaction is completed, except for morphology. Sperm is washed in a 15-mL conical centrifuge tube (3215, Costar) using HTF with 15% preovulatory serum (one part sperm, three parts medium), centrifuged for 10 minutes at 800 X g, and then the supernatant is discarded and a second identical washing is performed with 5 minutes of centrifugation. Supernatant is discarded again and 0.4 to 0.8 ml of the same medium is added to the pellet followed by incubation at 37 C for 40 minutes in a 5% CO2 atmosphere. The supernatant is retrieved and 1 to 2 fll of that sperm preparation is added to 4 to 5 fll of 10% polyvinyl pyrrolidone (mol wt 360,000; Sigma Chemical Corporation) and placed in the center of a Petri dish (Falcon 1006; Becton-Dickinson, Lincoln Park, NJ) and surrounded by eight 5-fLL droplets of injection medium (HTF-HEPES buffer and 10% bovine serum albumin fraction V [BSA; Sigma Chemical Corporation]) containing one oocyte each. They then are covered with 3 ml of light mineral oil. Preparation of sperm obtained by microepididymal sperm aspiration and tes- 574 Abdelmassih et al. Female age and ICSI Fertility and Sterility

3 ticular sperm aspiration is not included and will be the subject of another report. The ICSI procedure then is carried out on the heated stage at 37 C, (Fryer A 50; Fryer Co., Inc., Huntley, IL) of an inverted microscope at x400 magnification using a Hoffman Modulation Contrast System (Modulation Optics Inc., Greenvale, NY). The microscope is equipped with a Hitachi MOS VK C 150 color video camera (Hitachi Ltd, Tokyo, Japan) and a video monitor (Aiwa Br 8000, Tokyo, Japan) by which the procedure can be followed. The microscope is equipped with two coarse positioning manipulators (3D motor driven coarse control manipulator MM-188; Narishige, Tokyo, Japan) and two, three-dimensional hydraulic remote-control micromanipulators (Joystick Hydraulic Micromanipulator MO-188; Narishige). The pipets are fitted to a tool holder and are connected by Teflon tubing (CT-1; Narishige) to an 800-J.LL type microinjector CIM-6; Narishige), one for the holding and one for the injection pipet. A single sperm is rendered immobilized by crushing the tail. The tail is aspirated first from the central droplet into the tip of the injection pipet. One oocyte is stabilized using pre-established negative pressure exerted at the tip ofthe holding pipet. The polar body is held at 12 or 6 o'clock depending on the orientation of the bevel of the injection pipet in such a way that the injection is performed near the theoretical position of the spindle. The sperm is injected into the ooplasm in 1 to 2 pl of injection medium (HTF-HEPES buffer with 0.5% BSA fraction V; Sigma Chemical Corporation), making sure that the zona pellucida and the oolemma have been pierced during the injection by aspirating a small amount of ooplasm into the injection pipet. The 00- cytes then are washed twice with HTF medium with 15% of preovulatory serum. Mter the injection, the oocytes are incubated in a 25-J.LL droplet ofthe same medium and covered with mineral oil for 16 to 18 hours. The oocytes then are observed for the presence or absence of pronuclei and polar bodies. Fertilization is considered normal when two clearly distinct pronuclei containing nucleoli are present. If a single pronucleus is observed, a second evaluation is carried out approximately 4 hours later. If fertilization by these criteria has not occurred after 20 hours, reinjection can be considered. When the presence of three pronuclei is detected, the oocyte is discarded. Oocytes containing two pronuclei are put together in 1 ml of the same medium in a Petri dish (3260, Costar) and incubated until the next morning. The pre-embryos are observed at 42 hours after injection and classified according to the criteria proposed by the Brussels group (5). Pre-embryos having >50% fragmentation are not transferred. At the time of transfer (42 to 44 hours after injection) three to four pre-embryos are loaded in 1 ml of 100% preovulatory serum into a transfer catheter (K-TFTC-4020 W A Cook Australia PTY Ltd) and transferred into the uterine cavity via the cervix. Supernumerary pre-embryos with <20% fragmentation are cryopreserved. The patient rests in bed for 1.5 hours after transfer and then is sent home with light rest instruction for 24 hours. Patient Monitoring The luteal phase is supplemented with daily injections of 100 mg 1M P in oil (Schein Pharmaceutical Inc., Florham Park, NJ) until a pregnancy test is performed 13 days after pre-embryo transfer. In patients with positive,b-hcg levels, weekly monitoring using,b-hcg, E 2, and P serum levels and a 6-week transvaginal US examination is performed; the presence of an intrauterine pregnancy as well as of single or multiple sacs, fetal pole, and cardiac activity is ascertained. Pregnancies are monitored by the patient's obstetrician and the final outcome is reported to our clinic by the obstetrician and the patient. Statistical Analysis A Student's t-test was used to compare means. A X 2 test was used to compare percentages. RESULTS The total number of oocytes obtained in the 114 cycles was 1,181: 954 preovulatory (80.5%), 174 immature (14.7%), and 53 (4.5%) atretic, for a mean of 10.3 oocytes and 8.3 preovulatory oocytes per cycle. Eight hundred forty-seven preovulatory oocytes were injected and 107 were donated. Six hundred fifty-nine injected oocytes showed normal fertilization (78%) and 85.5% ofthem (564/659) went ahead and cleaved normally. Table 1 Results According to Age Age of patients (y) <30 31 to to 40 >41 No. of patients No. of cycles No. of preovulatory oocytes No. of preovulatory oocytes fertilized Fertilization rate (%) Abdelmassih et al. Female age and lcsl 575

4 Table 2 Results According to Age Age of patients (y) No. of patients with pre-embryo transfer Total number of pre-embryos transferred Mean number of pre-embryos per transfer No. of ongoing and term pregnancies* Total number of gestational sacs Total implantation ratet (%) < (41.6) to to 40 > (41.4) 4 (16.6) * Values in parentheses are percentages per transfer. t Means: women <35 years of age, 15.0%; women >36 years of age, 8.9%. A total of 107 of 114 cycles went to transfer (93.8%). Four hundred fifty-five pre-embryos were transferred (x = 4.3 pre-embryos per transfer) and 109 either were frozen or showed abnormal cleavage with very high fragmentation and were discarded. A total of 49 clinical pregnancies were established (43% total pregnancy rate per cycle and 45.8% per transfer). Thirty-six of these pregnancies are ongoing normally or went to term at the time of this writing (31.5% term and ongoing pregnancy rate per cycle and 33.6% per transfer). Twelve miscarriages were identified (24.5%) and one was an ectopic pregnancy. When the age of the patient population was considered (Tables 1 and 2), the transfer and fertilization rates and the mean number of pre-embryos transferred showed no significant differences in the age groups. The ongoing pregnancy rate dropped very significantly after the age of 35 years and, in the six patients treated after the age of 41 years, no pregnancies were established. An ongoing pregnancy rate per transfer of 41.5% was recorded in patients under 35 years of age, but it was only 13.3% after age 36 years (P < 0.01). The overall implantation rate was 13.4%, but again it showed a drop after age 36 years from 15% to 8.9%. This trend, although not significant because of the small population involved, may explain, at least in part, the results obtained in the two older age brackets. DISCUSSION After the first publications of unsuccessful attempts at ICSI (Veeck LL, Oehninger S, Acosta AA, Muasher SJ, abstract) (6) two main problems were considered to explain the failures: the trauma inflicted to the oocyte cytoarchitecture by the injecting pipet and the fluid incorporated into the ooplasm, and the sperm quality, which under different conditions had demonstrated a very poor fertilizing ability (7). The early results obtained by the group in Brussels rapidly dispelled both concerns. Better technology demonstrated that injury was not a problem and that the use of sperm of extremely poor quality produced an almost identical success rate and therefore was not a limiting factor. In conventional IVF, age has proved to be a very important prognostic indicator of final outcome, having a critical impact on abortion, implantation, and term pregnancy rates (8). Therefore, we decided to look into this prognostic indicator in our clinical material because almost no publication has addressed that issue in terms oficsi results at the time of this writing. The review of our material clearly demonstrated significant impairment of pregnancy rate and possibly of implantation rate in women over the age of 35 years, with extremely poor results in patients older than the age of 41 years. This brings up once again the fact that female fertility is very much age related and that the problem of oocyte and/or endometrial aging is very relevant in these cases. Even extreme cases of male infertility can be overcome by ICSI, to the point that it has been suggested that in 1995 there is almost no case of testicular insufficiency that cannot be solved by this procedure (Silber SJ, Nagy Z, Liu JJ, Tournaye H, Lissens W, Devroey P, van Steirteghem AC, abstract) but it is clear that the female, even when considered normal, also has a very important role to play. The oocyte quality, if age related, does not reflect in the fertilization and cleavage rates when ICSI is performed and therefore the transfer rate and the mean number of embryos per transfer are unaffected. All of these important pieces of information need to be transmitted to the patient for a more adequate informed consent. REFERENCES 1. Palermo G, Joris H, Devroey P, Van Steirleghem AC. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 1992;340): Abdelmassih et al. Female age and lcsl Fertility and Sterility

5 2. Palermo G, Joris H, Derde M-P, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steri11993; 59: World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction, 3rd ed. New York: Cambridge University Press, 1993: Palermo G, Joris H, Devroey P, Van Steirteghem AC. Induction of acrosome reaction in human spermatozoa used for subzonal insemination. Hum Reprod 1992;7: Camus M, Van den Abbeel E, Van Waesberghe L, Wisanto A, Devroey P, Van Steirteghem AC. Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertil Steril 1989;51: Ng S-C, Bongso A, Ratnam SS. Microinjection of human 00- cytes: a technique for severe oligo-astheno-terato-zoospermia. Fertil Steril 1991;56: Kruger TF, Acosta AA, Simons KF, Swanson RJ, Matta J-F, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49: Padilla SL, Garcia JE. Effect of maternal age and number of in vitro fertilization procedures on pregnancy outcome. Fertil Steril 1989;52: Abdelmassih et a1. Female age and ICSI 577