Prima facie, the IVM protocol utilized provided acceptable clinical and ongoing pregnancy rates that are comparable to those seen in routine IVF [17]. The results are also comparable to recent high IVM results reported in recent publications [18-20]. Based on the 58% clinical pregnancy rate in this series, a randomized trial, with an 80% power to detect a 10% difference in clinical pregnancy rates with a probability less than 5% that it wrongly detects a difference when one is not present and a 20% chance that it fails to detect a difference that truly exists, would require a total of 780 subjects. Such a study would be difficult to perform in a country where advanced reproductive technology is both expensive and often self-pay
IVM derived oocytes are suspected of lacking cytoplasmic competence compared to IVF [3,6,7,9,20]. Although competence has a complex biochemical basis [9,22,23], its endpoint is most easily seen in an oocyte’s ability to mature, fertilize, cleave, become a blastocyst, become a clinical pregnancy, and result in a live birth. The notion that the IVM process leads to a lack of oocyte competence was most strongly supported by earlier treatment series reflecting a lower pregnancy rate, a lower fertilization rate, and a lower implantation rate in patient cycles using IVM compared to patient cycles using IVF [1,2,5,7,14]. Pregnancy and live birth rates reflect the capacity of the “best” embryos (as selected by an individual embryologist) produced in an ART cycle and not on the entire cohort of oocytes retrieved and their resulting embryos. This paper’s findings suggest that a subset of oocytes, the oocytes that become the best blastocysts, was not compromised by the IVM process. Such oocytes were also adequately numerous to result in a pregnancy rate comparable to IVF.
Since this was not a controlled study and since there is significant variation between IVF programs, to better understand the results, an appropriate comparison group from our patient population was sought that had undergone IVF treatment using an approach as close to the above IVM protocol as possible. Our program cryopreserved all embryos in patients who were believed to be at high risk for OHSS or who experienced OHSS symptoms prior to a day 5 transfer. Preceding the program’s adoption of routine use of an agonist trigger for high risk patients, this was a relatively common occurrence. Such patients were also likely to have high antral follicle counts and frequently had polycystic ovarian syndrome. We identified 60 such patients who had all their embryos frozen because of OHSS concerns and who underwent IVF in the same monthly cycle groups as the IVM patients in this study. All patients in this comparison group utilized ISCI, shared the same environmental conditions, were exposed to the same IVF products (media, oil, protein) after ICSI, used the same FET protocol, and used the same approach to embryo transfer (double embryo transfer) as the IVM group. In spite of the similarities between this group and the IVM group, since the IVF group was selected based on different criteria, using it as a comparison group was a significant limitation of this study. Table 1 is a presentation of both the demographics and the outcomes of this group of IVF patients to those in this IVM series. To reiterate, this group of IVF patients was selected post hoc and is not a control group for this study. The rate of blastulation and the number of oocytes and blastocysts produced were significantly higher in this IVF group. Other clinical measures of oocyte competence were not a priori different.
The three major differences, between the IVM protocol used here from the more common approaches that have been used for IVM in the past, may be contributors to the increased competence compared to much of the IVM literature. The first major difference was the use of letrozole directed at small antral follicles to potentially enhance oocyte competence. Growth of preantral follicles and small antral follicles is driven by androgens. Androgens, during early antral follicle development, promote granulosa cell mitosis, increase FSH receptors, increase FSH sensitivity, and decrease follicle atresia [11,12]. The use of letrozole directly addresses the issue of potential decreased competence of IVM derived oocytes. It is only in the late antral follicle phase (9 to 10 mm), that follicles become FSH dependent for their growth. Theoretically, the impact of androgens on granulosa cell mitosis also aids in oocyte retrieval by creating more larger antral follicles available for easier IVM aspiration [24].
The second difference was in retrieval technique, which utilized the Steiner-Tan needle to minimize the time that the oocytes were out of their ovarian follicles and delivered to the laboratory. This needle differed from traditional IVF needles because its functional dead space was about 0.06 ml; whereas, a traditional needle (with tubing) has dead space of about 1.5 ml [25]. It differed from a traditional double-lumen IVF flushing needle, which used separate channels for flushing and aspirating, and therefore, had dead space similar to a single channel needle (unless copious flushing were done). A commonly used retrieval technique for IVM had been to aspirate several antral follicles into a needle (single or double lumen) before removing the needle from the patient and then rinsing the needle to provide an aspirate for the laboratory to evaluate [26]. In our hands, using that technique, oocytes might remain in the needle and tubing for up to five minutes. Aspirates could be evaluated only after using a cell culture screen to filter out clots and debris. The oocyte identification process was more time consuming and tedious than routine oocyte identification after IVF. Crane et al, found that a retrieval to incubation interval of more than four minutes in IVF was associated with diminished fertilization [27]. With traditional IVM retrieval techniques (without copious flushing), it would be unusual to not expose some oocytes to residence in the room temperature portion of the collecting system for at least 5 minutes. This aspect of IVM differs from IVF, where oocytes are usually immediately aspirated into a collection tube and quickly identified by the embryologist. The fluid dilution which occurs with the use of the Steiner-Tan needle and intentional vigorous flushing also make oocyte identification with IVM more like oocyte identification with IVF and thus easier [28].
The third difference in this protocol from most protocols used for IVM was the avoidance of fresh transfers. Immature oocyte retrieval occurred before a patient’s follicles were able to produce much endogenous estradiol, which potentially led to varied environments for endometrial development prior to fresh IVM transfer [15,16]. Some published IVM cycle series have reported a miscarriage rate as high as 50% [5]. Early studies evaluating methods to artificially develop endometria that were optimally receptive to embryo implantation focused on the duration of adequate estrogen exposure [14,29]. In this series, the potential problems of low estrogen and short duration of estrogen were avoided by using FET. Possibly as a result, the miscarriage loss rate in the first trimester (14.3%) was similar to routine IVF.
Recently, an international group has popularized a different approach to IVM which adds a pre-maturation laboratory step to IVM. This approach used oocyte priming with FSH without hCG. Retrieved oocytes underwent a 24 hour “pre-maturation” incubation with C-type natriuretic peptide to delay meiosis followed by maturation enhanced by incubation with amphiregulin. With this approach, FET was also utilized for embryo transfer. This protocol was found to increase oocyte competence, as reflected in a higher maturation rate, an increased percentage of good quality embryos, and a higher clinical pregnancy rate, compared to oocytes obtained using more traditional approaches to IVM [18]. Vuoing, et al, also used this protocol in a randomized study demonstrating equivalence of IVF and IVM with FSH only for priming and day 3 FETs [19]. It may be valuable to combine the ideas used in this paper’s protocol together with the pre-maturation step employed by Vuong and others.
The combined use of FSH and hCG during an IVM cycle enabled some oocytes to become metaphase II (MII) on the day of retrieval [8], which potentially confounds interpretation of the results. In the present study, the average number of oocytes which were MII on the day of retrieval was 0.71 per IVM cycle. There were 11 patients who had at least one MII oocyte on the day of retrieval. The blastulation rate resulting from the oocytes that were MII on the day of retrieval was 72.4% (compared to 42% for the IVM subgroup excluding these cycles). The subgroup consisting of these eleven women with at least one oocyte having early maturation had an implantation rate of 35.2% and a clinical pregnancy rate of 57.1% (compared to 34.2% and 57.9% for the full IVM group). Thus, there is little difference in clinical outcome in patients who did or did not have a mature oocyte on the day of IVM retrieval when the entire cohort of oocytes is considered. Those patients, who produce a mature oocyte at IVM retrieval, may benefit from their likelihood of having more excess blastocysts for use in subsequent FET cycles.