Premature Ovarian Failure

 

Background
Intracytoplasmic sperm injection (ICSI) is a component of infertility treatment often employed when conventional in vitro fertilization is unlikely to be successful. Despite good clinical results with ICSI, the procedure is typically associated with degeneration of a significant percentage (approximately 10%) of the treated oocytes. The cause of this degeneration remains unclear. Speculation that damage caused by oocyte compression during the injection procedure may be responsible has led to the development of a novel technique known as laser-assisted ICSI. This procedure involves drilling a small hole through the zona pellucida with a laser prior to sperm injection. Preliminary studies have suggested that laser-assisted ICSI may dramatically reduce oocyte degeneration rates. The objective of this study was to examine whether the reported benefits of laser-assisted ICSI could be verified on a larger, less-selected group of patients.

Methods
Oocytes retrieved from 59 patients scheduled for ICSI were randomly divided into equal treatment and control groups. Oocytes in the treatment group were inseminated by laser-assisted ICSI, while oocytes in the control group were inseminated by conventional ICSI. Outcome variables (oocyte fertilization and degeneration, embryo cell numbers and fragmentation on days 2 and 3, and compaction and blastocyst formation rates) were compared between treatment and control groups by paired-sample t-test. Subgroup analysis was performed according to zona pellucida and oolemma breakage patterns.

Results
No significant differences between treatment and control groups were observed for any of the measured outcome variables. However, fragile zonae pellucidae were associated with significantly poorer embryo quality, and fragile oolemmas that broke easily upon insertion of the injection needle were associated with a significantly higher oocyte degeneration rate. Nevertheless, there were also no between-treatment differences in clinical outcomes within these patient subpopulations.

Conclusion
Contrary to previous reports based on smaller sample sizes, the results of this study suggest that there is no benefit of laser-assisted ICSI, either for the general population of ICSI patients, or for patients prone to zona pellucida or oolemma fragility.
 

Background
Intracytoplasmic sperm injection (ICSI), a method of in vitro fertilization (IVF) in which a single sperm is introduced directly to the cytoplasm of a mature oocyte, has revolutionized the treatment of male factor infertility. Since the first reported pregnancies and births in 1992 [1], ICSI has become the treatment of choice for male factor infertility. In addition to this original indication, the use of ICSI has since expanded to a variety of other applications, such as when fertilization is reduced or absent following conventional in vitro insemination, when using cryopreserved sperm, and when preimplantation genetic diagnosis for monogenetic diseases is planned. Some authors have even advocated the generalized use of ICSI for all patients undergoing IVF [2]. Intracytoplasmic sperm injection was used in more than half of all fresh ART procedures using patients' own eggs performed in the United States during 2002 [3]. Given this widespread use of ICSI, any modifications that improve clinical outcomes of this procedure could benefit a large segment of the patient population.

Since its inception, ICSI has been performed by the mechanical penetration of the zona pellucida and the oolemma by a glass needle through which the sperm is injected into the cytoplasm. Although the effectiveness of this procedure has been clearly demonstrated [1,4,5], ICSI is typically associated with oocyte degeneration rates ranging from 5% to 19% [1,4,6-9]. The reasons for this oocyte degeneration are unclear. Observations of the zona pellucida of oocytes by scanning electron microscopy revealed no zona fragmentation during the procedure, and demonstrated that the injection hole closes immediately after the needle is withdrawn, with the injection site being nearly undetectable 15 minutes later [10]. Thus, zona damage does not appear to be the cause of ICSI induced oocyte degeneration. The mechanical compression and distortion of the oocyte that commonly occurs, to varying degrees, during injection is another potential source of damage leading to oocyte degeneration. Some oocytes may be more prone to damage from the ICSI procedure than others. For example, it has been reported that post-ICSI oocyte degeneration rates are significantly higher for oocytes with a fragile oolemma that breaks easily upon insertion of the injection needle than for other oocytes [6,7].

A novel approach of pre-drilling a hole through the zona pellucida with a laser prior to ICSI has recently been reported [11-14]. This alternative approach, called laser-assisted ICSI, allows the insertion of the sperm injection needle with less distortion of the oocyte and may therefore be less traumatic. Rienzi et al. [11] first reported a pregnancy using laser-assisted ICSI in a couple with four previous conventional ICSI failures and poor oocyte survival. They noted minimal oocyte deformation with the use of laser-assisted ICSI, and survival of 8 of the 13 metaphase II oocytes retrieved and injected. Two small randomized studies of patients with a previous history of high rates of oocyte degeneration (>20%) following ICSI, or who produced oocytes with fragile oolemmas, yielded dramatic statistically significant reductions in oocyte degeneration rates [12,15] (0.5% versus 16% and 2% versus 14%, respectively) and improved embryo quality [12] with the use of laser-assisted ICSI compared to conventional ICSI. Similar benefits have been claimed for piezoelectric ICSI [16].

The objective of this study was to evaluate the benefits of laser-assisted ICSI among unselected patients undergoing in vitro fertilization with ICSI. We describe results of the largest randomized trial examining laser-assisted ICSI yet reported, including comparisons of oocyte fertilization and degeneration rates, cell number and fragmentation on culture days 2 and 3, and compaction and blastocyst formation rates.

Methods
Patients were enrolled and treated following discussion of the study and informed written consent between March and September of 2004. The Western Institutional Review Board (WIRB) approved the study protocol (WIRB Pro. Nr. 20040267).

Indications for ICSI were diagnosis of moderate to severe male factor infertility or a history of poor conventional insemination. All patients undergoing IVF with ICSI at Shady Grove Fertility Reproductive Science Center during the study period were eligible for participation in the study, limited to one cycle per patient. The only exclusion criterion was retrieval of fewer than six mature oocytes. Mature (Metaphase II, or MII) oocytes from each participating patient were divided into two equal groups. Half of each oocyte cohort was assigned to the treatment group while the other half was assigned to the control group. Group assignments were allocated according to a computer-generated randomized series, were kept in sealed envelopes and were unknown to embryologists until after division of the oocyte cohorts. Treatment assignments were coded on embryo culture dishes, but effective blinding of embryologists recording fertilization outcomes and embryo development was not possible, because the hole in the zona produced by the laser was sometimes visible.

Laser-assisted ICSI was performed on oocytes assigned to the treatment group using a 1.48 micrometer wavelength infrared laser (Zilos Laser System, Hamilton Thorne Research, Beverly, MA). We attempted to replicate the procedure described in previous reports [11,12] as closely as possible. A small diameter (5–6 micrometer) channel was drilled through most of the thickness of the zona pellucida using 3 to 5 low energy pulses at less than two microseconds pulse duration; the sperm injection needle was then passed through this channel. Conventional ICSI was performed according to standard protocols [17] without laser pre-drilling. Within individual patients, a single embryologist performed ICSI on all mature oocytes in both treatment groups. For oocytes inseminated by conventional ICSI, the zona pellucida was graded as normal, fragile, or difficult according to breakage characteristics when the injection needle was inserted. Oolemma breakage patterns (normal, fragile, or difficult) were recorded for oocytes in both treatment groups. Subgroup analyses were planned based on these zona and oolemma characteristics.

The primary outcome measure was the percentage of oocytes that degenerated following the ICSI procedure, as this was considered the most direct indicator of ICSI-induced damage. Degenerating oocytes were distinguished by dark necrotic cytoplasm and vacuolization at the time of examination for fertilization, 16–18 hours after ICSI, when the number of 2 pronucleus (2pn) oocytes was also recorded. Numbers of cells per embryo and degree of fragmentation (percentage of total embryo volume) were recorded on days 2 and 3 of in vitro culture. Embryos were transferred to patients on either day 3 or day 5 depending on the number and quality of embryos available. Non-transferred embryos were monitored through day 7 for compaction and blastocyst formation.

Total numbers of oocytes and embryos are reported in the tables along with the per-cycle means for clinical outcomes. However, it must be emphasized that valid analysis of treatment effects for this experimental design is based on the per-cycle means, not outcomes per oocyte or embryo. It is well-known that oocyte and embryo characteristics are much more variable among patients than within oocyte cohorts. Thus, comparison of outcomes per oocyte or per embryo violates the assumption of independent observations. In this respect, the conclusions of the first randomized study [12] may be questioned because the results were evaluated by X² analysis using individual oocytes, rather than patients, as the unit of analysis. The statistical test used in the other prior randomized study [15] was not reported, but p-values are also consistent with per-oocyte X² analysis of the data. Valid estimates of either the size of, or the p-value for, the treatment effects from these two previous studies are not available.

Paired-sample two-tailed t-tests were used to compare oocyte fertilization and degeneration rates, numbers of cells and degree of fragmentation on days 2 and 3, compaction and blastocyst formation rates, and numbers of embryos transferred between conventional and laser-assisted ICSI treatments. The enrollment goal was set at 60 patients, approximately twice the size of the only previous randomized study published at the time [12]. Power analysis was conducted according to Bausell and Li [18]. Previous examination of oocyte degeneration among patients undergoing ICSI at our center [19] provided estimates of the expected mean (10%) and standard deviation (14%) for oocyte degeneration rates. Based on these parameters and assuming no correlation between treatment and control outcomes within patients (i.e. r = 0), this sample size would provide a minimum of 80% power to distinguish a reduction in oocyte degeneration with laser-assisted ICSI of 7.3% (i.e. 2.7% versus 10%), less than half the difference reported by Abdelmassih et al. [12]. Any correlation between the treatment and control groups within patients would increase statistical power. For example, a moderate correlation of r = 0.4 would result in 95% power to distinguish the above difference, and 80% power to distinguish a reduction in oocyte degeneration of 5.6%.

Results
A total of 80 candidates were enrolled following informed written consent. Twenty-one of these patients were subsequently excluded from the study because fewer than six mature oocytes were retrieved. From the remaining 59 patients, 775 mature oocytes were retrieved and inseminated in accordance with the experimental design.

Patient age ranged from 22 to 43 years (mean = 34.3 ± 4.0 SD). The majority of couples were diagnosed with male factor infertility alone (34/59, 58%). Other diagnoses included endometriosis (n = 4), tubal factor (n = 3), PCO (n = 3), diminished ovarian reserve (n = 2), ovulation disorder (n = 2), and unexplained infertility (n = 7). The remaining four couples were diagnosed with a combination of both male and female factor infertility. The overall mean fertilization rate was 71.1%, with a mean oocyte degeneration rate of 12.0%. Mean embryo cell number was 3.0 on day 2 and 5.6 on day 3. Mean degree of embryo fragmentation was 7.6% on day 2 and 9.8% on day 3. Embryos were transferred to 44 patients at the cleavage stage on day 3 (mean = 2.5 embryos per transfer), and to 14 patients at the blastocyst stage on day 5 or 6 (mean = 1.9 embryos per transfer). In only one cycle was no transfer performed due to poor embryo quality. There were 26 pregnancies (44.8% per transfer) with 34 implantations (24.5% per embryo), based on ultrasound detection of fetal cardiac activity five to six weeks after embryo transfer, resulting in 20 live births and 3 spontaneous abortions, with three pregnancies ongoing at last report.

Internal server error