1 Department of Gynecology and 2 Department of Perinatology, Bialystok Medical University, Sklodowskiej 24A, Bialystok 15276, 3 CryobankInfertility Center, Bialystok, Poland and 4 Infertility Center, Department of Obstetrics and Gynecology, Ghent University Hospital, Belgium
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Abstract |
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Key words: ICSI/IVF/pregnancy/reinsemination/total fertilization failure
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Introduction |
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From the beginning of IVF, various procedures to salvage the lost cycle have been attempted. Reinsemination of the unfertilized oocytes 24 h after oocyte retrieval has been tried. (Boldt et al., 1987; Calderon et al., 1993
). After the advent of micromanipulation techniques, partial zona dissection (PZD), subzonal insemination (SUZI) and finally ICSI were introduced to rescue oocytes after unsuccessful IVF (Malter et al., 1989
; Nagy et al., 1993
; Van Steirteghem et al., 1993
; Wiker et al., 1993
; Imoedemhe and Sigue, 1994
; Lundin et al., 1996; Morton et al., 1997
; Bussen et al., 1997
; Loong et al., 1997
; Yuzpe et al., 2000
). Although pregnancies have been reported after rescue of failed fertilization, most reports were relatively inconclusive. A large prospective study on the success of rescue ICSI in terms of pregnancy is still lacking.
The aim of our study was to compare the results of cycles with TFF in which rescue ICSI was performed with a matched control group of ICSI cycles, both in terms of fertilization and pregnancy outcome and to answer the question whether rescue ICSI is worth trying.
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Materials and methods |
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All couples in the IVF group met standard criteria for admission to IVF, including normal semen motility and density as assessed by published criteria (World Health Organization, 1992). The matched control group (group II) was selected retrospectively and consisted of 280 couples undergoing an ICSI procedure during the same period. Seventy patients from each year of the study period were matched to group I patients according to age, type of stimulation and number of oocytes retrieved.
ICSI was performed when <50 000 sperm with type a progressive motility (World Health Organization 1992) and good morphology were present in the whole ejaculate. The overall proportion of ICSI versus IVF cycles was ~62% in our IVF centre at that time.
Stimulation protocol and oocyte retrieval
Normogonadotrophic patients with normal-sized ovaries on ultrasound were treated either with a GnRH agonist and hMG in a short protocol (group I, 71; group II, 160) or clomiphene citrate followed by hMG (group I, 14; group II, 35). In the flare-up protocol, buserelin (Suprefact®, Hoechst) was used in a dose of 0.4 ml per 24 h s.c. starting from day 12 of the cycle and hMG (Humegon®, Organon or Pergonal®, Serono) or FSH (Metrodin®, Serono) was added on day 3 of the cycle, starting with a dose of 150 IU clomiphene citrate (Clostilbegyt®, CibaGeigy) was given at a dose of 100 mg per day from day 3 to day 7 of the cycle and the stimulation was continued with hMG, two ampoules per day from day 8 onwards.
Patients with polycystic ovaries (confirmed by ultrasonography; group I, 35; group II, 85) were treated with a long protocol, using either a short-acting GnRH agonist (Suprefact) or a long-acting GnRH agonist (Decapeptyl®, BeaufourtIpsen or Decapeptyl depot®, Ferring). The GnRH agonist was administered on day 21 of the previous menstrual cycle. After desensitization of the pituitary gland was confirmed, FSH and/or hMG were given in individually adapted doses, as a rule starting with 150 IU per day (Metrodin® or Pergonal®; Ares-Serono; or Humegon®, Organon). Ovulation was triggered with hCG (Pregnyl®; Organon Inc., Canada), 10 000 IU i.m., when at least three follicles had a diameter of 19 mm and serum estradiol concentration was 1000 pg/ml. Oocytes were retrieved 35 h after hCG administration by transvaginal ultrasound-guided follicular puncture under sedation with i.v. pethidine and diazepam.
Sperm preparation
Semen was collected by masturbation after 35 days of sexual abstinence. The ejaculates were allowed to liquefy (at 37°C) and examined within 0.5 h after collection. The evaluation according to published criteria (World Health Organization, 1992) was carried out under a light microscope. After examination sperm samples were cryopreserved for potential later use. For ICSI, either fresh or cryopreserved sperm were used. In three cases, motile sperm retrieved from the IVF incubation plate were used for rescue ICSI.
The semen preparation was performed as follows. An appropriate volume of fresh semen or cryopreserved sample, after 15 min of thawing in room temperature, was diluted with 5 ml of Earles medium. After centrifugation (10 min at 800 g, room temperature) the supernatant was removed and replaced with another 5 ml of Earles medium. After a second centrifugation, the supernatant was once again removed and the pellet was overlaid with 1 ml Earles medium and incubated (37°C, 5% CO2 in air) to perform separation by swim-up. For ICSI both supernatant and pellet, if needed, were used.
Assessment of fertilization and embryonic development
In IVF cases, cumuluscorona radiataoocyte complexes were inseminated in 4-well plates (Falcon) with 50 000150 000 motile spermin Ménézo BII medium and placed in the CO2 incubator (Heraeus, Germany) at a temperature of 37°C in 5% CO2 in air. Fertilization was checked 1820 h after insemination. Absence of pronuclei and a second polar body in the perivitelline space were proof that fertilization had not occurred. This was further confirmed after granulosa cells and sperm bound to the zona pellucida were removed enzymatically (hyaluronidase) and mechanically.
Unfertilized metaphase II oocytes were microinjected as described previously (Van Steirteghem et al., 1993), immediately after failed fertilization was observed and oocytes were checked for survival and fertilization 24 h later (Nagy et al., 1994
). Fertilization was considered to be normal when two individual or fragmented polar bodies and two pronuclei were present. The quality of embryos was evaluated 48 h after ICSI. Embryos were graded as good (<20% anucleate fragments) fair (2050% anucleate fragments) and poor (>50% anucleate fragments). Embryos with <50% fragmentation were eligible for transfer.
A maximum of three embryos was transferred into the uterus on day 3 after ICSI by using a Wallace (UK), Labotect (Labotect GmbH, Germany) or Frydman (OSI, France) catheter. On the day of hCG injection, a sham transfer with the Frydman catheter was performed to probe the cervical canal. The Wallace and Frydman catheters were used randomly for non-complicated transfers, the Labotec catheters were used for difficult transfers. The distribution of these three types of catheters was equal in group I and group II.
Supernumerary embryos were cultured with Vero cells and cryopreserved if they reached the blastocyst stage. The luteal phase was supported with intravaginally administered micronized progesterone, 600 mg daily (Uterogestan®, Piette, Belgium).
Pregnancy
Pregnancy was confirmed when rising serum ß-hCG concentrations were found on at least two successive occasions. Clinical pregnancy was defined by the presence of a gestational sac and fetal cardiac activity by ultrasound at ~7 weeks of pregnancy.
Statistical analysis
Statistical evaluation was performed using Students t-test, 2-test, Fishers exact test or the MannWhitney test as appropriate. Differences were considered significant at P < 0.05.
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Results |
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Discussion |
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Because fertilization at IVF is considered the ultimate proof of the fertilizing capacity of the sperm, there is an intuitive tendency to conversely assume that failure of fertilization is due to some sperm defect. When the sperm is normal according to the classical criteria, an as yet undisclosed sperm defect is presumed to be present (Mahadevan and Trounson, 1984; Jeulin et al., 1986
; Liu and Baker, 2000
). Poor or absent binding of sperm to the zona pellucida, for instance, is readily ascribed to abnormalities of the sperm (Liu et al., 1989a
,b
). There are, however, no firm data to substantiate this hypothesis. Pregnancies either spontaneously or in subsequent IVF cycles do occur in patients with previous unexplained failure of fertilization, which suggests that occult sperm deficiencies, if they exist, are transient. There are, however, several other possible reasons for oocytes not being fertilized after IVF when the sperm is apparently normal. Bedford et al. demonstrated that inability of apparently competent sperm to penetrate the zona is often associated with ooplasmic anomalies such as refractile bodies, extra groups of chromosomes, chromatin rings or masses as well as one or more pronuclei with one or no polar bodies (Bedford et al., 1993). Also further downstream, a number of defects in the fertilization process such as sperm fusion with oolemma, nuclear decondensation or formation of the male pronucleus and failed oocyte activation have been reported (Dozortsev et al., 1994
; Flaherty et al., 1995
; Palermo et al., 1997
). Besides a disordered spermoocyte interaction, there are a number of developmental defects of the oocyte which can impede fertilization and/or the reproductive outcome. Spontaneous cytoplasmatic activation resulting in premature cortical granule loss and zona pellucida hardening (Ducibella et al., 1995
) or cytoplasmic degeneration, manifested by refractile bodies, can affect fertilization (Serhal et al., 1997
). Arrest or asynchrony of cytoplasmic maturation can impact on the reproductive outcome of fertilized oocytes (Kubiak, 1989
).
Faced with the frustrating experience of all oocytes remaining unfertilized the day after in-vitro insemination, all kind of attempts have been made to rescue the cycle by reinsemination or micromanipulation of the oocytes. Lundin et al. were the first to report pregnancies by microinjection of unfertilized IVF oocytes (Lundin et al., 1996). Other centres also reported successes with pregnancy rates ranging from 6.9 to 20.7% (Bussen et al., 1997; Morton et al., 1997
; Yuzpe et al., 2000
). However, in some cases, mixed transfers of IVF-ICSI embryos were performed (Table IV
). Not all reports, however, are positive and a number of authors have reported negative results similar to ours and advise against the practice of rescue ICSI (Chen et al., 1995
; Park et al., 2000
).
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The normal pregnancy rate obtained in the patients with previously failed fertilization who were subsequently treated by ICSI proves that in the majority of cases, failure of fertilization is not due to an intrinsic and permanent defect of either sperm or oocytes.
In conclusion, rescue ICSI, although resulting in fertilization, gives very poor results in terms of pregnancy. Prolonged incubation of oocytes presumably affects the developmental capacity of the embryo. Based on our experience, rescue ICSI of unfertilized oocytes after IVF does not seem to be worthwhile.
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Notes |
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References |
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Submitted on March 8, 2002; accepted on May 21, 2002.