The effect of insemination/injection time on the results of IVF and ICSI

Marleen Jacobs1, Annette M. Stolwijk2 and Alex M. M. Wetzels1,3

1 Fertility Laboratory, Department of Obstetrics and Gynaecology and 2 Department of Medical Affairs, University Medical Center Nijmegen, Nijmegen, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to investigate whether a pre-incubation time between oocyte retrieval and insemination or injection had any effect on the success rate of IVF or intracytoplasmic sperm injection (ICSI). Based on previously published data, many laboratories retain a time interval of several hours between oocyte retrieval and insemination/injection. In our setting, insemination and injection times are dependent only on the laboratory workload. METHODS: Totals of 881 IVF and 432 ICSI cycles performed between 1997 and 1999 were analysed retrospectively. Oocyte retrieval occurred 36 h after human chorionic gonadotrophin administration, and insemination or injection took place 1–7 or 0.5–8 h after oocyte retrieval respectively. RESULTS: No statistically significant differences were found between these time periods and outcome of IVF and ICSI with respect to fertilization rate, embryo quality, implantation rate, abortion and ongoing pregnancy rates, except for the abortion rate after IVF. As this finding may be due to chance and no differences were found in the ongoing pregnancy rates, this finding was considered to be of less importance. CONCLUSIONS: If laboratory control and efficiency demands early insemination or injection, it could be performed without reservation.

Key words: ICSI/IVF/outcome/pre-incubation/timing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In IVF or intracytoplasmic sperm injection (ICSI) programmes, oocytes are usually retrieved in hyperstimulated cycles at about 36 h after human chorionic gonadotrophin (HCG) injection (Elder and Dale, 2000Go). In a stimulated cycle, ovulation would occur at about 37 h after the HCG surge (Edwards and Steptoe, 1975Go). For this reason, many centres carrying out IVF and ICSI use a defined time interval after oocyte retrieval in which insemination or injection must occur.

At present, there is some discussion regarding whether injection in ICSI cycles can be performed shortly after oocyte retrieval. It has been reported (Rienzi et al., 1998Go) that a pre-incubation period of at least 3 h between oocyte retrieval and injection in ICSI cycles could improve the fertilization rate and the embryo quality. In contrast, two other studies reported no statistically significant differences in the ICSI fertilization rate (Van de Velde et al., 1998Go) and pregnancy rates (Yanagida et al., 1998Go) in relation to the timing of injection.

With respect to IVF, only older reports detail the effects of pre-incubation time on IVF outcome. Indeed, conflicting results were found: in some studies the best IVF results were obtained if the oocytes were inseminated after a pre-incubation period of 3–5.5 h after retrieval (Trounson et al., 1982Go; Harisson et al., 1988Go; Khan et al., 1989Go), while in another study there was no effect (Fisch et al.1989Go). In these early years, techniques such as laparoscopic oocyte retrieval and ovarian stimulation with clomiphene citrate were used which might have influenced the outcomes.

In theory, some problems can be related to insemination/injection time. According to one report (Kubiak, 1989Go), pre-ovulatory mouse oocytes are not fully mature, although a first polar body is present (so-called cytoplasmic immaturity), leading to lower fertilization rates. Likewise, others (Calafell et al., 1991Go) reported that a pre-incubation period can decrease the premature chromosome condensation rate in immature (human) oocytes. However, prolonged culture of (mouse) oocytes can lead to zona hardening, increased parthenogenetic activation and impaired embryonic development (Fukuda et al., 1992Go).

In our laboratory, the time period between oocyte retrieval and insemination/injection has been shown during recent years to be dependent only on the laboratory workload, and ranges from 30 min to 8 h. Thus, in a retrospective study, we may add to the discussion of whether it is necessary to pre-incubate human ICSI oocytes before injection, and hence draw conclusions regarding a possible role for these time-related problems in human ICSI oocytes. As ICSI might bypass some critical steps in the fertilization process, the results of the procedure are not automatically valid for standard IVF. For this reason, the effects of the timing of insemination in IVF were also evaluated; this can be seen as an update of older studies and as an effort to obtain a more complete view on this subject.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Data were used from patients who underwent IVF or ICSI treatment in the University Medical Centre Nijmegen (UMCN), The Netherlands, between January 1997 and September 1999. Excluded were cycles in which no oocyte retrieval was performed, in which more than two embryos were transferred, in which the time between oocyte retrieval and embryo transfer was not equal to 3 days, and in which cryopreserved embryos were transferred. Only cycles that were performed completely in the UMCN were included. Donor and frozen gametes were not used. Five IVF cycles and seven ICSI cycles were excluded because of the absence of (in case of ICSI, mature) oocytes after follicular aspiration. Moreover, only those cycles were included where the time period between oocyte retrieval and insemination or injection was known. This resulted in an inclusion of 881 IVF cycles and 432 ICSI cycles from a total of 797 patients. The patient selection criteria for ICSI were: (i) a total motile sperm count <1x106, and/or <0.5x106 after sperm preparation over a density gradient; and (ii) two previous fertilization failures at IVF.

The patients received the so-called `long protocol' for ovarian stimulation using gonadotrophin-releasing hormone agonist (Decapeptyl®; Ferring, Hoofddorp, The Netherlands ) and human menopausal gonadotrophin (Humegon®; Organon, Oss, The Netherlands ) or recombinant FSH (Puregon®; Organon ). Ovulation was triggered by using HCG (Pregnyl®; Organon ). The oocytes were collected transvaginally under ultrasound guidance, at 36 h after HCG administration.

Laboratory routine
In our laboratory, the daily routine began with the determination of fertilization and selection of embryos to be transferred between 08:30 and 10:00. Subsequently, oocytes were retrieved and semen was prepared, followed by embryo transfer. Finally, IVF-insemination and culture medium preparations were carried out. Sperm injections were performed throughout the day, depending on the availability of personnel.

Oocyte and sperm handling
After oocyte collection, abundant cumulus cells were dissected and the oocyte–cumulus complexes were washed and incubated in IVF medium consisting of human tubal fluid medium (,Quinn et al.1985; Bio Whittaker, Verviers, Belgium ) supplemented with 10% pasteurized plasma solution (gpo; CLB, Amsterdam, The Netherlands). For ICSI, the oocytes were treated with hyaluronidase (Medi-cult, Jyllinge, Denmark) and denuded with a capillary pipette before injection was performed. All oocytes were incubated (37°C, 5% CO2 in air ) in IVF medium.

Freshly ejaculated semen was mixed with 5 ml IVF medium, layered onto an 80% Pure-Sperm (Nidacon International, Gothenberg, Sweden) gradient and centrifuged at 600xg for 20 min. The pellet was washed twice with IVF medium. In the case of IVF, oocytes were inseminated with spermatozoa to a concentration of ~105 motile spermatozoa/ml in 2 ml of IVF medium. ICSI was performed according to a published method (Van Steirteghem et al., 1995Go); only motile spermatozoa that appeared morphologically normal (as determined under ICSI magnification) were injected. The time of insemination or injection depended only on the workload in the laboratory.

On the morning after insemination and injection, the oocytes were scored for the presence of 0, 1, 2 or >=3 pronuclei (PN). The fertilization ratio was defined as the fraction of 2 PN oocytes of the total number of inseminated/injected oocytes. Subsequently, all oocytes were transferred to fresh 100 µl droplets of IVF medium under mineral oil (Allegiance, Zutphen, The Netherlands), taking into account that 0, 1 and >=3 PN oocytes were cultured separately from 2 PN oocytes. On day 3 after oocyte retrieval (64–72 h post insemination), all embryos were scored for cell number and embryo quality score on a scale from 1 to 4 (Steer et al., 1992Go): grade 4 (perfect), no fragmentation; grade 3 (good), <10% fragmentation; grade 2 (fair), 10–50% fragmentation; and grade 1 (poor), >50% of the embryo fragmented. The one or two embryos with the highest cell number and embryo quality score were selected for embryo transfer. Transfer of embryos was performed using a Wallace catheter with or without an obturator (Laprolan, Beuningen, The Netherlands); in cases where the Wallace catheter was not stiff enough, a Frydman catheter (Allegiance) was used. Supernumary embryos of good quality (grade 4 or 3) were cryopreserved.

Luteal phase and pregnancy
The luteal phase was supported with progesterone (Progestan®; Organon ). A pregnancy test was performed on day 15 after embryo transfer, and at about week 10 of pregnancy an ongoing pregnancy (one or more fetal sacs with heart activity) was confirmed by ultrasonography.

Statistical analysis
Statistical analyses were performed for IVF cycles and for ICSI cycles separately. If the number of patients within each hourly time period, between oocyte retrieval and insemination or injection, was small, the groups were combined. This resulted in five groups for IVF (1–2, 2–3, 3–4, 4–5 and >=5 h after oocyte retrieval), and four groups for ICSI (0–2, 2–3, 3–4 and >=4 h after oocyte retrieval). In order to study whether there was any association between the pre-incubation time and cell number, embryo quality score and pregnancy outcome, only cycles in which an embryo transfer was performed were used. For the univariate analyses, the Kruskal–Wallis test was used to test for differences in percentages of fertilization, cell numbers and embryo quality scores between time periods. Cell number was defined as the number of blastomeres of the transferred embryo; in the case of two transferred embryos, the number of blastomeres was only counted in the embryo with the highest quality score. A comparable method was used to define embryo quality score. The {chi}2-test was used to test differences in positive pregnancy tests, abortions and ongoing pregnancies between time periods.

Logistic regression analysis was applied to adjust for potential confounding effects. Hence, the percentage of fertilization were dichotomized into <50% and >=50%, the cell number on day 3 into <8 and >=8 (assuming that an embryo should reach the 8-cell stage in vitro at ~60 h post insemination; Weima, 1996), and the embryo quality score into <4 and 4. The cycle number was included as a potential confounding factor. In addition, the number of fertilized oocytes was considered to be a potential confounder in the other outcomes. Furthermore, if the relationship of the time between oocyte retrieval and injection was studied in ICSI cycles, the person who performed the injection was also included as a potential confounding factor. Results were presented as odds ratios (OR) and likelihood ratio tests. An OR of 1 indicates that there are no differences between groups. Likelihood ratio tests were used to study whether the time period explained any variation in the outcomes. In the first instance, all IVF cycles and all ICSI cycles were used to study whether there was any effect of timing of insemination or injection on the outcome of IVF or ICSI respectively. Second, as one couple could have undergone more than one cycle, and therefore the cycles under study were not independent, a check was made to determine whether the results changed if only the first IVF cycle was used, and the first ICSI cycle of each couple during the studied period (for the individual patient, this might be the first to eighth cycle). If a couple underwent both an IVF cycle and an ICSI cycle during this period, data from their first IVF cycle during this period, as well as data from their first ICSI cycle during this period, were used for these analyses.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to investigate the effect of time between oocyte retrieval and insemination or injection on the outcome of IVF or ICSI respectively, data from 881 IVF cycles and 432 ICSI cycles were used. Only cycles in which at least one oocyte was retrieved (in the case of ICSI, a mature oocyte) were included. The time between oocyte retrieval and insemination or injection ranged from 1–7 h in the case of IVF, and from 0.5–8 h in the case of ICSI. The mean (±SD) and range of the women's age was 33.6 ± 4.1 years (23–42) in IVF, and 32.8 ± 4.1 years (21–44) in ICSI. The maximum duration of subfertility was 17 years (mean 4.0 ± 2.3) in IVF; for ICSI, the maximum duration of infertility was 16.5 years (mean 4.3 ± 2.4).

Cycle characteristics and results of the included IVF and ICSI cycles during the study period are shown in Table IGo. Total fertilization failure occurred in 151 (96%) of the 158 IVF cycles, and in six (75%) of the eight ICSI cycles without embryo transfer. With respect to fertilization rate, cell number and embryo quality score, no significant differences were found between the different insemination/injection time intervals (Tables II and IIIGoGo).


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Table I. Characteristics of all IVF and ICSI cycles
 

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Table II. Success of IVF cycles per time period between punction and insemination (n = 881 )
 

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Table III. Success of ICSI cycles per time period between punction and insemination (n = 432 )
 
In IVF, the implantation rate was 14.7% (15 of 102 embryo transfers) if only one embryo was transferred; however, if two embryos were transferred, both were implanted in 8.4% of cases (52/620), while only one embryo was implanted in 20.0% of cases (124/620). The outcome was unknown for one embryo transfer. In ICSI, the implantation rate was 10.8% (4/37 embryo transfers) if only one embryo was transferred; however, if two embryos were transferred, both were implanted in 9.9% of cases (38/385), while only one embryo was implanted in 24.7% of cases (95/385). Again, the outcome of one embryo transfer was unknown. For IVF as well as for ICSI, there was no association between time period and implantation rate (100% versus 50% or 0%) (data not presented).

No statistically significant differences were observed between the IVF time periods with regard to positive pregnancy tests and ongoing pregnancies, although the rate of positive pregnancy tests in the first IVF time period appeared to be somewhat higher (Table IIGo). In contrast, the percentage of abortions was significantly higher (P = 0.005 ) in the first time period, leading to comparable ongoing pregnancies in all time intervals.

In the case of ICSI, no statistically significant differences were observed between the time periods with regard to positive pregnancy tests, ongoing pregnancies and abortions (Table IIIGo). The percentage of positive pregnancy tests ranged from 33.3% per embryo transfer in the fourth hour to 44.7% per embryo transfer in the third hour. The ongoing pregnancies ranged from 28.2 to 34.9% per embryo transfer in the fourth hour and first two hours respectively.

The unadjusted and adjusted OR for IVF and ICSI cycles are listed in Tables IV and VGoGo respectively. For these ratios, the last time period was the reference time. The OR changed only slightly after adjustment for confounding factors, indicating that there were hardly any confounding effects. In concordance with the results of the univariate analyses (Tables II and IIIGoGo), the results of the unadjusted and adjusted logistic regression showed that there was no relationship between the time period and the success of IVF or ICSI. There was one exception here, however, the time between oocyte retrieval and insemination being associated with abortion. If only data of the first cycle per couple in this time period were analysed, 574 IVF cycles and 293 ICSI cycles remained, and comparable results were found (data not shown).


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Table IV. Unadjusted and adjusted odds ratios (OR) between success of IVF and time period between punction and insemination (n = 881 )
 

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Table V. Unadjusted and adjusted odds ratios (OR) between success of ICSI and time period between punction and injection (n = 432 )
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to investigate whether there was any effect of the duration of pre-incubation time (prior to insemination or injection) on the success rate of IVF or ICSI. No significant differences were found with regard to fertilization rate, embryo quality implantation rates and pregnancy rates. An association with abortion was only found after IVF (P = 0.01 ), but this finding cannot be explained.

At present, different opinions exist concerning the maintenance of pre-incubation time of oocytes in IVF and ICSI (see Introduction). With respect to ICSI, only one group have found that the fertilization rate and embryo quality were reduced if injection occurred within 3 h after oocyte retrieval (Rienzi et al., 1998Go). The discrepancy of these authors' results with those of others (Van de Velde et al.1998Go; Yanagida et al.1998Go) and of the current study might be explained by the fact that Rienzi et al. considered all oocytes/embryos from each patient as statistically independent observations, which is incorrect. Moreover, the number of patients in their study was small. Therefore, we consider that these early results (Rienzi et al.1998Go) might be misleading. As there were no obvious differences in stimulation protocols and culture conditions between these three studies and the current study, it is concluded that the timing of injection is not relevant to the outcome of the ICSI procedure.

With regard to IVF, a defined pre-incubation time before insemination does not seem to be relevant with the techniques and drugs used at present. However, any effects of very short pre-incubation intervals cannot be excluded as there were no cycles with a pre-incubation time <1 h.

One remarkable observation was the high number of positive pregnancy tests in IVF, followed by a significantly higher abortion rate, if insemination occurred within 2 h of oocyte retrieval. An explanation for this phenomenon—which was not seen in the ICSI group—cannot be provided, and hence we consider that this result is due to `chance'. However, it will be interesting to follow especially this group in the future, with regard to the `take-home baby rate' and the development of the children.

The development of children might be important as it was reported that pregnancies from aged oocytes in mice can lead to growth-retarded pups, delayed development of the righting reflex and higher spontaneous motor activity and emotionality of F1 offspring (Tarín et al., 1999Go). It is not known whether cytoplasmic immaturity can lead to comparable problems in the offspring. Zona hardening and impaired embryonic development was reported after prolonged culture of mature mouse oocytes in vitro (Fukuda et al., 1992Go), while others (Saito et al., 1993Go) mentioned changes in the alignment of chromosomes which lead to decreased fertility.

According to others (Kubiak, 1989Go; Eppig et al., 1994Go), nuclear and cytoplasmic maturation are not necessarily coupled, and oocytes gradually develop the capacity for activation during the MII arrest. A lower fertilization rate was reported (Kubiak, 1989Go) in mouse IVF if the oocytes were inseminated just before onset of ovulation. No information was provided about the subsequent development of embryos and the performance of the offspring, however. Also in mice, the development of embryos was reported to be blocked during early cleavage if MII oocytes were retrieved from pre-pubertal mice (Eppig et al.1994Go). Because both phenomena (lower fertilization rate and embryonic arrest) were not observed in the current study, it is concluded that it is implausible that cytoplasmic immaturity is a factor of influence in human IVF and ICSI procedures. For the same reason (no lower fertilization rate), it is possible that premature chromosome condensation, as described by others (Schmiady et al., 1986Go; Calafell et al., 1991Go) does not occur to any major extent.

Based on the results of the current study and on the cited literature, it is concluded that the results of IVF/ICSI are independent of the timing of insemination/injection within a time interval of about 30 min to about 6 h. Besides the scientific interest of this finding, there are consequences for the logistics in the laboratory. The unimportance of insemination/injection time within these limits may lead to a higher grade of efficiency (and quality) in the IVF laboratory.


    Notes
 
3 To whom correspondence should be addressed at: Fertility Laboratory, Department of Obstetrics and Gynaecology,University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: a.wetzels{at}obgyn.azn.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 8, 2000; accepted on April 26, 2001.