The effect of in vitro maturation (IVM) protocol changes on measures of oocyte/embryo competence

Purpose: In vitro maturation (IVM) continues its evolution as new ideas are introduced with the objective of making the IVM procedure easier and more effective. This study combines ideas believed likely to improve the IVM outcome or make the IVM oocyte identi�cation process easier. Methods: A cohort of 45 women underwent an IVM cycle in which letrozole was used with the theoretical objective of improving the competence of small antral follicles, the oocyte aspiration technique was modi�ed to minimize the time between oocyte aspiration and oocyte identi�cation, and blastocysts were transferred during a subsequent cycle with controlled endometrial development. Results: Measures of oocyte competence used for these prospectively followed cycles were as follows: the maturation rate was 90.5%, the fertilization rate was 92.4%, the cleavage rate was 94.6%, the usable blastulation rate per zygote was 50.2%, and the implantation rate was 34.2%. In 7.9% of cycles initiated, there was a failure to produce blastocytes. Per transfer, the biochemical pregnancy rate was 63.2%, the clinical pregnancy rate was 55.3% and the ongoing/delivered pregnancy rate at the end of the �rst trimester was 47.4%. The miscarriage rate for clinical pregnancies in the �rst trimester was 14.3% and the ongoing twinning rate was 11.1%. Conclusion: Except for blastocyst production, the above modi�cations of IVM technology were suggestive of oocyte competence similar to conventional IVF. In particular, the clinically best blastocysts derived from IVM cycles appear to function as well as the best blastocysts derived from oocytes produced during a traditional IVF cycle. Ethics support: Baptist Health Institutional Review Board (IRB Number 18-49)


Introduction
The application of in vitro maturation (IVM) of human oocytes to in vitro fertilization and embryo transfer (IVF) has been successful in producing offspring for more than 30 years.Yet, compared to IVF, IVM is not commonly utilized [1,2,3].Even for the subset of patients for which IVM has been most successful, women with high antral follicle counts, IVM is only used in rare patients.Initially, there was much enthusiasm about a procedure that did not use high doses of gonadotropins, eliminated the risk of uncontrolled ovarian hyperstimulation, and was otherwise gentler for patients than conventional IVF [4,5,6].Some programs which have tried to incorporate IVM, have expressed concern that compared to conventional IVF, IVM was more time consuming, oocyte identi cation at retrieval was technically different, and the reported clinical pregnancy rates seemed to vary widely between programs, and were often low compared to IVF [1,5,7].
An adjunct preceding an IVM retrieval, called "priming", is aimed at trying to optimize the competence of the oocytes so that they may become embryos which develop, implant and become babies.Most commonly, these adjunctive treatments included an injection of hCG preceding retrieval, treatment with 450 IU of FSH split over three days, or this same FSH treatment together with an injection of hCG [8,9].An additional method of priming, which is not commonly used, is treatment with letrozole during the time period that antral follicles are expected to be small [10].The use of letrozole at the beginning of an IVM cycle is reasonable since normal follicle development from the preantral stage to small antral stage has been shown to be dependent on androgen availability [11,12].
The traditional approach to IVM retrieval involves aspirating several follicles and because of the small volume of those follicles, removing the needle periodically to ush out oocytes contained within the needle collection set [6].This approach to oocyte aspiration potentially exposes the oocytes to opportunities for prolonged cooling while in the needle or the tubing.The amount of cooling would vary with the protocol for aspiration, such as, how frequently the needle was removed for ushing, the type of needle used, and the speed of the surgeon.It is possible to rapidly ush the oocyte out of the needle and into a collection tube and, theoretically, decrease the potential for oocyte cooling in the needle or collecting tubing.With good needle placement, most antral follicles can be used as a receptacle for ush media.With repeated lling and emptying, enough uid can be moved through the needle to ensure that uid in the needle and collecting system tubing., potentially containing oocytes, was moved into the collection tube, which can then be passed to the laboratory [13].
The IVM literature reports a wide range of miscarriage rates after pregnancy using IVM with about half of early programs, which published a miscarriage rate, reporting a clinical pregnancy loss rate of greater than 25% [5,7,14].This may have been due to the widely differing endometrial environments into which fresh embryos were transferred [14][15][16].The literature forming the basis for endometrial preparation prior to donor embryo transfer in IVF, and subsequently, frozen embryo transfer (FET), is at odds with the endocrine environments commonly created during fresh transfer IVM cycles which may have low estrogen levels and/or evidence of poor endometrial response [14][15][16].IVM transfer could be made more uniform by cryopreserving embryos and transferring them back to the patient during a routine FET cycle.
Such an approach has recently been used in a program with low pregnancy loss results [16], but is not uniformly used across all IVM programs [6,7].
The objective of this paper was to evaluate the consequences of using a de ned protocol that utilizes all three of the forestated modi cations of more common approaches to IVM; namely, priming with letrozole during the small antral follicle stage, using a retrieval technique focused on minimizing the time from oocyte pickup to oocyte identi cation, and avoiding fresh embryo transfer by utilizing vitri ed blastocysts in a routine FET cycle.

Materials And Methods
Patients were either self-referred or selected because of prior poor response to letrozole/clomiphene citrate and/or FSH.Patients were also required to be under age 39, have an antral follicle count greater than 25, and an AMH level greater than 3.5 ng/ml.These criteria were chosen to aid staff in referring patients to the study and to limit the confounding impact of the increase in aneuploidy with advanced age.Participants also were required to have ovaries easily assessable by transvaginal ultrasound and have a BMI under 40 (original protocol 35: changed to better recruit from a PCO population).All patients considering IVM were counseled that IVM should be viewed as an experimental procedure, consistent with ASRM recommendations at the time [7].They were also informed that the procedure was to be done under an experimental protocol monitored by Baptist Health Institutional Review Board (IRB Number 18-49).This protocol was also enrolled in clinicaltrials.gov(NCT04149496).The proposal submitted to the IRB included the use of the Steiner-Tan pseudo double lumen needle, which had not been FDA approved for use in the United States.A consultation with the FDA con rmed that the needle could be used under an IRB protocol with appropriate labeling and as long as the use had no commercial component (under 21 CFR 812 (m)).
Patients were treated with letrozole 2.5 mg for ve to seven days at the time follicle were expected to be maximally responsive to androgens [12].FSH (Gonal-F, Serono, Merck, Darmstadt, Germany) 25 to 75 IU was added for three to ve days overlapping the use of letrozole by three days with the objective of providing FSH to the larger antral follicles.HCG (Ovidrel, Serono) was given 38 hours prior to the planned retrieval.Operating room and embryologist availability limited IVM retrievals to a speci c three days during each cycle month.The variation in medication administration was used to try to optimize patients to t into the limited available retrieval opportunities.Most patients received 2.5 mg of letrozole for ve days and 50 IU of FSH for four days starting on the third day of letrozole.Cycles were canceled if no antral follicles with diameters greater than 5 mm were present or if the patient had follicles greater than 14 mm in diameter prior to available retrieval days.The objectives of this protocol were to increase androgen availability to small antral follicles (< 6-7 mm) and augment FSH availability to larger antral follicles (> 8-10 mm).In a program with greater exibility than ours, letrozole (2.5 mg) would be started on or before cycle day 3 and low dose FSH (25-50 IU) would be added when antral follicles were su ciently large.Letrozole would be stopped after a 2 day overlap of FSH.FSH would be stopped and hCG given with the plan to move to retrieval with a su cient cohort of 8-12 mm follicles.
Oocyte retrievals utilized the Steiner-Tan pseudo double lumen 17 gauge needle (IVFETFLEX, Ganz, Austria).This was a 17-gauge needle with a plastic outer sheath which carried the ush media so that it entered the needle about 6 cm from the distal end and so that ush media moved both into the distal and proximal parts of the needle (Fig. 1).The technique used was to aspirate an antral follicle and, if needle placement was adequate, repeatedly rapidly ush the follicle with a total of four to ten ml of phosphate buffered saline (PBS) while continuing to aspirate.The objective was to clear the needle and tubing of each potentially retrieved oocyte as quickly as possible.
Oocytes obtained after IVM were identi ed as rapidly as possible to minimize their exposure to a nonoptimal environment.Identi cation of oocytes was done by decanting collection tubes lled with follicular uid and PBS onto a petri dish for examination under a dissecting microscope, as done for routine IVF.Approximately four hours after collection, cumulus cells were minimally disrupted to determine the maturity of each oocyte.

Results
Forty-ve IVM retrievals were performed with data recorded prospectively.Three IVM retrieval cycles were excluded from this study, because they violated the protocol.Two of these were excluded because they were IVM "rescue" cycles and used an IVF gonadotropin protocol rather than the above IVM protocol (both patients delivered singletons from these IVM rescue cycles).The last excluded cycle was performed on a 40 year-old women, who had required IVM in the past to achieve pregnancy.Her cycle resulted in a biochemical pregnancy.Four cycles did not have a transfer.In one cycle, the husband could not produce sperm.In three cycles, no blastocysts were produced.Data from these four cycles were included in the 42 IVM cycles reported on below (except for calculations involving blastocysts and pregnancy rates).
The average age of these patients was 30 ± 4.

Discussion
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][19][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 di cult 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 re ecting 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 re ect 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 ndings 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 signi cant 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 identi ed 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 signi cant 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 signi cantly 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 rst 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 doublelumen IVF ushing needle, which used separate channels for ushing and aspirating, and therefore, had dead space similar to a single channel needle (unless copious ushing 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 ve minutes.Aspirates could be evaluated only after using a cell culture screen to lter out clots and debris.The oocyte identi cation process was more time consuming and tedious than routine oocyte identi cation 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 ushing), 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 identi ed by the embryologist.The uid dilution which occurs with the use of the Steiner-Tan needle and intentional vigorous ushing also make oocyte identi cation with IVM more like oocyte identi cation 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 arti cially 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 rst trimester (14.3%) was similar to routine IVF.
Recently, an international group has popularized a different approach to IVM which adds a prematuration 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 re ected 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 bene t from their likelihood of having more excess blastocysts for use in subsequent FET cycles.

Conclusions
The modi cations of IVM technology to include letrozole use for early antral follicle development, rapid clearing of the collection system to minimize the time between the oocyte leaving the ovary and its identi cation by the embryologist, and the use of FET to control and standardize embryo transfer for IVM derived blastocysts results in acceptable indicators of oocyte competence di cult to distinguish from IVF derived oocytes.More blastocysts are created from women with a high antral follicle count using IVF than using IVM, suggesting the full cohort of IVM derived oocytes is less competent than the cohort of IVF derived oocytes.However, the best IVM derived blastocysts appear to function as well as the best IVF derived blastocysts.Hopefully, the ideas used in this IVM series can be combined with other new approaches to advance the evolution of IVM technique.

Funding
The authors declare that no grants, or other support were received during the preparation of this manuscript.
IVM protocol (n = 38 The primary difference between the Steiner-Tan needle and other IVF oocyte collection needles is the two holes indicated by the arrow above.The polyethylene tubing sheath, which extends from the left of these holes to the top of the needle, has been removed.Flush uid travels through this tubing surrounding the needle and enters these holes to pass into the needle.Some of the uid goes through the short needle segment, located to the right of these holes, to ush the follicle, and some uid goes up the needle toward the collection tube.The uid moving toward the collection tube clears the needle of its contents.
Aspirating while ushing increases the amount of uid that clears the needle.
deactivated serum, obtained from the patient, under oil in a 5% CO 2 /O 2 environment at 36.5 o C. To maximize assessment of the ability of oocytes to mature in vitro, all oocytes were observed for 60 hours post retrieval for maturity with checks in approximately 8 to 12 hour intervals.Mature oocytes were then incubated for two hours in continuous single culture media (Irvine Scienti c/FujiFilm, Santa Ana, CA) with 5% synthetic serum (Cooper Surgical ART-3011) before being fertilized using ICSI.Zygotes were incubated in continuous single culture media with 5% synthetic serum and Irvine culture media (90164) under the same environmental conditions for a maximum of six days.Blastocysts were vitri ed (Irvine freeze kit 90133-SO or 90188) after grading and stored until needed for transfer.The patients subsequently underwent routine frozen embryo transfer (FET) after blastocyst warming (Irvine thaw kit 90137-SO or 90183).The practice protocol for all FET cycles in this IVF program was to transfer two blastocysts if they were available and if it was acceptable to the patient.More than 90% of our patients choose to transfer two embryos.
Immature cumulus oophorus complexes were incubated in a Nunn/Falcon 4-well dishes of IVM media (Sage, Cooper Surgical Medical Devices) with 75 mIU FSH/ml and 20% complement 3, the average BMI was 29 ± 7.2, and the average AMH was 10.2 ± 7.3.The average number of oocytes retrieved was 9.24 ± 4.01 with 90.5% of them becoming mature during the IVM incubation.Of the mature oocytes, 92.4% fertilized after ICSI.Of the zygotes, 94.6% cleaved and 50.2% became transferrable blastocysts.The average number of blastocysts produced per patient was 4.18 ± 2.19.An average of 0.71 oocytes per cycle were mature on the day of retrieval.Eleven patients had at least one oocyte mature on the day of retrieval.For the rst FET cycle after the IVM cycle, the biochemical pregnancy rate per transfer was 65.8% and the clinical pregnancy (with cardiac motion) rate per transfer was 57.9% (clinical pregnancies based on having a sac on ultrasound: 60.5%).The twelve week ongoing or delivered pregnancy rate per transfer was 47.4%.The twin live birth rate was 11.1%.The implantation rate for this series was 34.2%.No blastocysts were produced in 7.9% of these IVM retrievals.There were no cases of OHSS.Three viable clinical pregnancies were lost during the rst trimester.After this rst FET cycle, 29 patients had 86 cryopreserved blastocysts remaining for future use.