Buckett, William M. MD, FRCOG; Chian, Ri-Cheng PhD; Holzer, Hananel MD; Dean, Nicola PhD; Usher, Robert MD, FRCP(C); Tan, Seang Lin MBBS, FRCOG
Immature oocyte retrieval and subsequent oocyte maturation in vitro (IVM) without any ovarian stimulation is a new development in assisted reproductive technology (ART). Many pregnancies have been reported, and IVM is successfully performed in many parts of the world.1–5
In vitro maturation gives the benefits of ovarian stimulation—namely, more oocytes—without the risks of ovarian stimulation. Ovarian hyperstimulation syndrome is a potentially life-threatening condition associated with ovarian stimulation. Severe ovarian hyperstimulation syndrome affects 1–2% of all women undergoing ART and up to 6% of women with polycystic ovary syndrome (PCOS) or ultrasound-only polycystic ovaries.6 The only way to avoid ovarian hyperstimulation syndrome is to avoid ovarian stimulation (Buckett WM, Chian RC, Tan SL. Can we eliminate severe ovarian hyperstimulation syndrome? Not completely [letter]. Hum Reprod 2005;20:2367). Any new development in assisted reproduction must be accompanied with data concerning congenital abnormality, obstetric outcome, and later developmental sequelae.
Early data concerning pregnancies resulting from IVM have been generally reassuring so far (Mikkelsen AL, Ravn SH, Lindenberg S. Evaluation of newborns delivered after in vitro maturation [abstract]. Hum Reprod 2003;8 suppl 1: xviii5; Buckett WM, Chian RC, Barrington K, Dean NL, Abdul-Jalil AK, Tan SL. Obstetric, neonatal and infant outcome in babies conceived by in-vitro maturation (IVM): initial five-year results 1998–2003 [abstract]. Fertil Steril 2004;82 suppl 2:S133).7 However, previous studies of pregnancy and child outcome after ART interventions have been complicated by methodological issues and differences in the reporting of malformations8,9 or in the control groups.10
Therefore, the objective of this study was to compare pregnancies and infants born after IVM, IVF, and ICSI with those of age- and parity-matched controls, all of whom were delivered at a single hospital where the same criteria were used for the diagnosis of congenital abnormality and obstetric outcome.
MATERIALS AND METHODS
Ethical committee approval was not sought for this specific study because it is a review of data already collected. Local institutional review board rules determine that approval from the institution’s Director of Professional Services is needed. This was granted.
Couples needing ART were treated with IVF or ICSI, as appropriate. The indication for IVM was a need for IVF or ICSI, in the presence of a high antral follicle count, PCOS or ultrasound-only polycystic ovaries.
All pregnancies delivered at the McGill University Health Centre after assisted reproductive treatments (namely, IVM, IVF, or ICSI) with a birth weight of at least 500 g at the time of confinement from January 1, 1998 to December 31, 2003 were reviewed. Congenital abnormality, gestational age at delivery, birth weight, Apgar scores, cord pH, growth restriction, pregnancy complications (such as preeclampsia or gestational diabetes), mode of delivery, and multiple pregnancy were recorded.
Data were obtained from the McGill Obstetric and Neonatal Database (MOND).11 Information was entered at the time of delivery and final data reviewed 12 months after delivery. All children were examined within 48 hours of delivery and all abnormalities classified in a standard manner.
All pregnancies after IVM, IVF, and ICSI were then matched one-to-one with those of age and parity matched controls who had conceived spontaneously. The controls were selected from the same database. Control pregnancies were selected for the same age and parity as the index pregnancies and from women who delivered in the same calendar month. If there were no matches within the same month, then they were matched within the same calendar year.
Clinical and laboratory aspects of the in vitro maturation procedure have been reported elsewhere.12 Briefly, immature oocytes were retrieved transvaginally under ultrasound guidance from unstimulated ovaries between days 9 and 11 after the start of menstruation for ovulatory women and between days 9 and 14 after an induced withdrawal bleed for anovulatory women. Ultrasound scans between days 2 and 4 of the cycle and 2 days before oocyte retrieval were performed to exclude an ovarian cyst or dominant follicle of more than 10 mm diameter. Women received 10,000 International Units of human chorionic gonadotropin 36 hours before immature oocyte retrieval.13
The immature oocytes were incubated in TC-199 medium supplemented with inactivated maternal serum, pyruvic acid, and follicle-stimulating hormone+ luteinizing hormone for 24–48 hours until maturity. At metaphase II stage, all oocytes routinely underwent ICSI.
For endometrial preparation, patients received oral estradiol valerate, 6–12 mg daily, starting on the day of oocyte retrieval; the dosage depended on endometrial thickness. Luteal support was provided by 200 mg of intravaginal progesterone three times daily, from the day of oocyte maturation.
For IVF, most patients were treated with a modified long protocol14 or with the short or microdose flare protocols (Kelly SM, Pirwany IR, Phillips SJ, Tan SL. Microdose gonadotrophin-releasing hormone agonist [GnRH-a] flare protocol compared with standard long protocol GnRH-a for ovarian stimulation in patients undergoing in vitro fertilization [abstract]. Fertil Steril 2002;78 suppl 2:S234).15 After satisfactory pituitary suppression, ovarian stimulation was achieved with human menopausal gonadotropin or with recombinant follicle-stimulating hormone. Human chorionic gonadotropin was administered when there were at least three follicles with a mean diameter of at least 18 mm.
Transvaginal oocyte recovery was performed 36 hours after human chorionic gonadotropin administration. After retrieval, oocytes were collected into Vitrolife IVF media (Vitrolife Inc, Englewood, CO) and inseminated with 10 μL of prepared sperm, at a concentration of 5–7 M/mL motile sperm. Fertilization was confirmed 16–18 hours after insemination, and the embryo transfer was performed 2–3 days after oocyte retrieval, depending on the number and quality of the embryos available. The luteal phase was supplemented with 200 mg of micronized progesterone vaginally, three times daily, or alternatively with 50 mg intramuscular progesterone (in oil) daily from the day of oocyte retrieval.
For ICSI, ovarian stimulation, embryo transfer, number of embryos transferred, and the luteal phase support was the same as that for IVF. The mature (MII) oocytes were denuded of cumulus cells. Spermatozoa for ICSI were prepared by mini-Percoll separation (45% and 90% gradients) at 560g for 20 minutes. A single spermatozoon was injected into each metaphase II oocyte. Fertilization was assessed 18 hours after ICSI for the appearance of two distinct pronuclei.
Proportions and rates are presented by numerator and denominator and as percentages, normally distributed data by the mean and standard deviation, and nonnormally distributed data by the median and interquartile ranges. Normality was determined by the Shapiro-Wilk W test. The observed odds ratio was calculated for the retrospective risk of congenital abnormality. Categorical data were analyzed with the McNemar and exact (Liddel) tests for matched pairs. Analysis of variance using the Kruskal-Wallis test and Dunnet test was used for the multiple comparison procedures in pregnancy outcomes tables. All analyses were performed by StatsDirect (StatsDirect Ltd, Altrincham, Cheshire, UK).
A total of 432 children were born from 344 pregnancies after ART during the study period (IVM 55, IVF 217, ICSI 160). There were no stillbirths, neonatal deaths, or infant deaths in pregnancies conceived after IVM, IVF, or ICSI or among the age- and parity-matched spontaneously conceived control pregnancies. Within the institution as a whole, the total number of children born during the same period was 13,585, with a perinatal mortality rate of 7.4 per 1,000.
Mean maternal age in years was 33 for IVM pregnancies, 35 for IVF pregnancies, 34 for ICSI pregnancies, and 34 for control pregnancies. The median parity for all groups was 0. There were no statistically significant differences.
The congenital abnormalities from IVM, IVF, and ICSI are listed in Table 1. Of the major congenital abnormalities after IVM, the omphalocele required surgical correction, and the small ventriculo-septal defect needed no treatment.
Of the 350 infants born to the 344 age- and parity-matched spontaneously conceived controls, there were nine major congenital abnormalities (two hydronephroses, one Fallot’s tetralogy, one single ventricle, two atrio-septal defects, one hydrocephaly and intraventricular hemorrhage, and two trisomies), and 14 minor abnormalities.
The observed odds ratios (ORs) for any congenital abnormality were 1.42 (95% confidence interval [CI] 0.52–3.91) for IVM, 1.21 (95% CI 0.63–2.32) for IVF, and 1.69 (95% CI 0.88–3.26) for ICSI, respectively (Fig. 1). None of these were statistically significant.
Of the ART pregnancies, 267 (78%) were singleton pregnancies (IVM 31, IVF 132, ICSI 104), 66 (19%) were twin pregnancies (IVM 9, IVF 35, ICSI 22), and 11 (3%) were triplet pregnancies (IVM 2, IVF 5, ICSI 4). The triplet pregnancy rate was 5% (2 of 42) for IVM, 3% (5 of 172) for IVF, 3% (4 of 130) for ICSI, and 0% for controls (P<.001). The twin pregnancy rate was 21% (9 of 42) for IVM, 20% (35 of 172) for IVF, 17% (22 of 130) for ICSI, and 1.7% (6 of 344) for controls (P<.001). There were no significant differences in the multiple pregnancy rates between IVM, IVF, and ICSI pregnancies.
Overall cesarean delivery rates were higher after ART (IVM 21 of 42, IVF 78 of 172, ICSI 55 of 130) compared with controls (90 of 344). All triplet pregnancies were delivered by caesarean, and 71% (47 of 66) of all twin pregnancies were delivered by caesarean. Cesarean delivery rates were also higher in ART singleton pregnancies (IVM 39%, IVF 36%, ICSI 36%, controls 26%) compared with controls (P<.05) (Fig. 2).
Overall rates of instrumental delivery were 9.5% (4 of 42) after IVM, 11.0% (19 of 172) after IVF, and 3.8% (5 of 130) after ICSI. This compared with an instrumental delivery rate of 6.5% (22 of 338) in matched controls.
Mean birth weight of all infants—singletons and multiples—after ART was similar among all ART groups (IVM 2,813 g, IVF 2,826 g, ICSI 2,809 g), but was lower than that of controls (3,230 g) (P<.001). Similarly, there were no differences between the proportion of low birth weight infants (less than 2,500 g) and very low birth weight infants (less than 1,500 g) among the ART groups (IVM 33%, IVF 39%, ICSI 39%). However, these proportions were lower than in the controls (10%) (P <.001).
Birth weight in singleton ART pregnancies and controls are shown in Table 2. Mean birth weight of IVM infants (3,482 g) was higher than in control (3,260 g), IVF (3,209 g), and ICSI infants (3,163 g). Although the proportion of macrosomic infants was higher (10% compared with 4%), this did not reach statistical significance.
Mean gestational age at delivery of all infants—singletons and multiples—after ART was similar among all ART groups (IVM 36 weeks 6 days, IVF 37 weeks 2 days, ICSI 36 weeks 5 days), but was lower than that of controls (39 weeks 5 days) (P<.001). Similarly, among the ART groups, there were no significant differences between the proportion of premature deliveries before 37 weeks (IVM 38%, IVF 30%, ICSI 36%) or before 34 weeks (IVM 16%, IVF 10%, ICSI 18%). However, the proportion of deliveries that were premature was higher than that of controls (10% delivered at less than 37 weeks, 5% delivered before 34 weeks) (P<.001).
The results for gestational age at delivery in singleton ART pregnancies and controls are shown in Table 2. Although singleton IVF and ICSI infants had a lower mean gestational age at delivery (P<.001) and a higher proportion of deliveries before 37 weeks (P<.01), there was no significant difference in the proportion of deliveries before 34 weeks of gestation.
The median Apgar scores at 1 and 5 minutes, the proportion of infants with an Apgar score of 6 or less at 1 and 5 minutes, the mean cord pH, and the proportion of infants with mild acidosis (cord pH less than 7.2) or severe acidosis (cord pH less than 7.05) are shown in Table 3.
Weight for gestational age is determined by calculating the ratio between the birth weight at delivery and the standard norm for that gestation. These are shown in Table 3. Infants conceived after IVF had a slightly lower mean birth weight ratio (0.93) than controls (P<.01). The proportion of all infants with a birth weight ratio below the fifth percentile (birth weight ratio less than 0.8) was higher in infants conceived after IVF and ICSI compared with controls (P<.001).
The rate of gestational diabetes was slightly higher in pregnancies that resulted from IVM, at 17% (7 of 42) compared with IVF at 11% (19 of 172), ICSI at 10% (13 of 130), and controls at 7%. Similarly, the rate of preeclampsia was slightly higher after IVM, at 12% (5 of 42) compared with IVF at 5% (9 of 172), ICSI at 8% (11 of 130), and controls at 7% (25 of 344). However, these did not reach statistical significance.
This study shows that, so far, IVM is not associated with an increased risk of congenital abnormality compared with IVF, ICSI, or spontaneously conceived controls, although the numbers are still small. Nevertheless, the number of infants born after IVM is still greater than those reported in other series (Mikkelsen et al [abstract]; Buckett et al [abstract]).7 The strength of this paper, unlike that of other reported series, is that all infants born after IVM were delivered in the same center and underwent the same examinations and data collection as did the IVF, ICSI, and control infants. But because McGill is also a tertiary referral center for obstetrics, despite the fact that over 70% of deliveries are classed as low- risk pregnancies, there is still the risk of an ascertainment bias.
Over 100 infants have been delivered after IVM treatment at McGill, and no other major congenital malformations have been reported to us so far. However, many of these infants have been delivered at other hospitals in Québec or outside the province; therefore, accurate comparison with other ART-conceived pregnancies or spontaneously conceived controls in these externally delivered cases is impossible.
In the literature, to date, congenital abnormalities from a total of 103 infants born after IVM have been reported (Mikkelsen et al [abstract]; Buckett et al [abstract]).7 These abnormalities were cleft lip/palate (2), ventricular septal defect (1), and congenital dislocation of the hip (1). This gives a total rate of congenital abnormality similar to that in the general population and similar to that reported in this study. The abnormalities reported so far and those reported in this paper do not as yet show an increase of any particular abnormality.
In this study, all ART treatments have an increase in congenital abnormality. Although these findings do not reach statistical significance, they are consistent with increasing evidence from large ART-based studies, which show an increased prevalence of major malformations,16,17 chromosomal anomalies,18 and, more recently, imprinting disorders.19 Whether ART has a direct causative effect, however, is still the subject of much debate.20
This study reports the obstetric outcomes after IVM compared with other pregnancies conceived by IVF and IVF/ICSI and also with spontaneously conceived controls. The only previous study of obstetric outcome after IVM reported a series of 24 deliveries.21
The major obstetric complication associated with IVM, and also with IVF and ICSI, is the multiple pregnancy. This was the major reason that all ART pregnancies were associated with a lower mean gestational age at delivery compared with spontaneously conceived controls. Similarly, the mean birth weight was lower in all ART pregnancies compared with spontaneously conceived controls. However, there was no difference in the multiple pregnancy rate, gestational age at delivery, and birth weight when IVM was compared with IVF or with ICSI pregnancies.
When singleton pregnancies were compared, the mean gestational age was lower, the proportion of deliveries under 37 weeks was higher, and the incidence of growth restriction was higher in IVF and ICSI pregnancies. This is consistent with other data confirming these outcomes in singleton ART pregnancies.22
In vitro maturation singleton pregnancies were not associated with earlier delivery, and the mean birth weight is, in fact, higher than in controls. This could be a result of the patients having undergone IVM—usually those with PCOS or those with ultrasound-only polycystic ovaries. Gestational diabetes is associated with higher birth weight and macrosomia, and these findings may be a result of the inherent predisposing risk of polycystic ovaries/PCOS,23,24 rather than as a direct result of the treatment modality. Nevertheless, the numbers are still small and continuing surveillance of birth weight, macrosomia, and gestational diabetes is warranted.
In conclusion, when ART is indicated, IVM offers a reduction in the risks of ovarian stimulation. Based on current data, we have been unable to demonstrate any increased risk of congenital abnormality or perinatal outcome over that already accepted for IVF or ICSI. For an 80% power and a 5% alpha, with a probability of congenital abnormality in the population of 3% and a factor of 1.5, more than 1,000 IVM infants and more than 3,000 controls would be needed. The establishment of national and international registries for infants born after IVM, continued data collection and matched studies, and the results from ongoing child development longitudinal studies are essential as the use of IVM as a clinical treatment continues to expand.
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