Mosaic embryo transfer after oocyte in vitro maturation in combination with non-invasive prenatal testing (NIPT)—first report of a euploid live birth

Abstract

Purposes

The purpose of this study is to describe a healthy life birth after a mosaic embryo transfer in oocyte in vitro maturation (IVM).

Methods

Patient received minimal stimulation, starting on day 3 after menstrual period. No hCG trigger was administered. Oocyte retrieval was performed and oocytes were matured for 30 h. After denuding, mature oocytes were inseminated by ICSI. Embryos were cultured until blastocyst stage and biopsied.

Results

One euploid embryo after array comprehensive genome hybridization (aCGH) was diagnostic. However, the next-generation sequencing (NGS) re-analysis showed that embryo was a mosaic for chromosome 13 and 21. Nevertheless, pregnancy ultrasound scans and non-invasive prenatal test (NIPT–Verifi-Illumina) indicated a normal fetus development. Finally, a healthy baby was born after 38 weeks. Its weight was 4480 g, head circumference 36 cm, and total length of 51 cm. To confirm that the baby was chromosomically normal, an NGS test was performed in buccal cells, a normal profile was obtained.

Conclusions

Our finding confirmed that mosaic embryo transfer would bring a healthy offspring.

Introduction

The in vitro maturation (IVM) of human oocytes is a promising assisted reproduction technique. IVM is the maturation in culture of immature oocytes obtained from follicles between 2 to 10 mm [1]. During IVM, immature oocytes progress from a germinal vesicle (GV) until the extrusion of the first polar body [2]. Since the first pregnancy report after IVM of human oocytes by Cha [3], it has been suggested as a new clinical strategy. Despite the lack of a unique stimulation protocol and the variability in clinical outcomes [4], several strategies have been developed to maximize the maturation rates and clinical outcomes in selected patients [5–7].

IVM has been suggested as a new alternative in patients with polycystic ovarian syndrome (PCOS) for minimizing the risk of ovarian hyperstimulation syndrome (OHSS). Additionally, IVM is recommended for oncologic patients who require fertility preservation. This group of patients could be treated in follicular phase or luteal phase of menstrual cycle without postponing the oncologic treatment. Additionally, hormone levels (i.e., estrogens) would not be increased since IVM requires either none or minimal gonadotropin stimulation.

For many years, the clinical outcomes of IVM technique have been shown to be variable between IVF laboratories, showing pregnancy rates ranging from 0 [8] to 42% [9]. These findings indicate that hormone stimulation protocol for IVM needs standardization [10]; moreover, the culture media has not been changed substantially [4], and further prospective studies are needed to resolve the disagreement between fresh and frozen embryo transfer [11].

It is well-known that chromosome aneuploidy is a major cause of IVF failure, establishing that most embryo with aneuploidy do not implant or are miscarried during the first trimester of pregnancy [12]. Since the introduction of pre-implantation genetic diagnosis for aneuploidy screening (PGD-AS), the clinical outcomes and live birth rates have improved and reduced significantly the miscarriage rates [13, 14]. PGD-AS using array comprehensive genome hybridization (aCGH) has been replaced for the next-generation sequencing (NGS) technique which is capable to classify the embryos as euploidy, mosaic, or aneuploidy [15].

The first report using IVM with PGD-AS by FISH was performed by Ao et al. in 2006 [16]. This case demonstrated that sufficient number of embryos could be developed after IVM procedure followed by PGD-AS analysis in patients with PCO/PCOS. Likewise, other studies have mentioned successful pregnancies after IVM procedures where embryo biopsy was performed on day 3 and analyzed for fragile X syndrome and chromosome translocation [17, 18].

In the present report, we described an IVM treatment followed by trophoectoderm cell biopsy with aCGH analysis, NGS re-analysis, and a follow-up genetic assessment.

Case report

A 34-year-old woman was referred with a desire to conceive. She presented three negative results of intrauterine insemination (IUI). Two consecutive conventional IVF cycles were performed: the first cycle without success and the second cycle with a miscarriage during the first trimester. She only had one ovary, and it was polycystic on ultrasound scan; additionally, the patient presented high anti-mullerian hormone levels (15.4 ng/ml). Patient opted to undergo a minimal ovarian stimulation protocol for IVM technique followed by a PGD-AS analysis.

On day 3 of her menstrual cycle, minimal ovarian stimulation began with 150 IU per day of hp-HMG (Menopur, Ferring). After 3 days of stimulation, an ultrasound scan was performed and it showed that follicles were below than 10 mm [19]. Oocyte retrieval was performed 42 h after the last dose of gonadotropins [20]. Then, eight immature cumulus oocyte complexes were retrieved and cultured for 30 h in a rich media supplemented with FSH 75 mIU/ml and hCG 75 mIU/ml. After the IVM culture, the oocytes were denudated with hyaluronidase (LifeGlobal 80 IU/ml). Resulting in four (50%) mature oocytes, two (25%) metaphase I, one (12.5%) germinal vesicle, and one (12.5%) atretic oocyte.

Partner semen sample was obtained by masturbation; motile sperms were selected through density gradient of 95 and 45% of Isolate® (Irvine, US). Sperms were selected physiologically by using SpermSlow (Origio, Denmark) which consist in hyaluronic acid (HA) attached to the base of the culture dish for better sperm selection. The HA binding sites on sperm plasma membrane indicate sperm maturity, having better fertilization rates, and less sperm DNA fragmentation [21]. Mature oocytes underwent intracytoplasmic sperm injection.

The inseminated oocytes were cultured for 20 h in Global® Total fertilization media. Three zygotes were obtained after insemination and cultured in Global® Total. On day 4, three embryos developed and assisted hatching was performed with a laser (Lykos; Hamilton Thorne, Beverly, ME, USA) on multipulse function (pulse/s = 7; pulse duration = 150 μs); extended culture was done until day 6 where two fair quality blastocysts were biopsied (Fig. 1). Embryo grading was done according to SART classification [22].

Fig. 1.

a, b Blastocyst on day 5. c, d Blastocyst on day 6 before blastocyst biopsy. e The embryo showed on Fig. 1c was euploid and it was thawed for embryo transfer. f Euploid blastocyst was cultured for 2 h before embryo transfer

Biopsied cells were collected according to the genetic laboratory protocol. Blastocysts were vitrified within 1 h after TE biopsy using a Cryotech vitrification kit (Cryotec, Japan) following manufacture’s protocols. Each blastocyst was vitrified individually in an open system using Cryotech.

Biopsied trophoectoderm cells were processed for aCGH analysis and were sent to Reprogenetics Latinoamerica (Lima, Peru). Briefly, biopsies were collected in Eppendorf tubes containing 2 μl of non-sticking buffer and referred to the genetic laboratory. Samples were lysed and amplified using the SurePlex kit®. Amplified samples were processed following the protocol of BlueGnome Cyto-Chip. Microarray chips were scanned, analyzed, and quantified as previously described by Gutierrez-Mateo [23]. Copy number ratios were analyzed using Cyto-Chip algorithm fixed setting in BlueFuse software (BlueGnome). aCGH results shown that embryo showed in Fig. 1a, c was considered euploid while embryo showed in Fig. 1b, d was aneuploid for chromosome X (Fig. 2).

Fig. 2.

a aCGH profile from the euploid embryo. b NGS re-analysis showed that aCGH embryo was mosaic for chromosomes 13 and 21. c aCGH profile from aneuploidy embryo. d NGS re-analysis confirmed the aneuploidy and showed a mosaicism for chromosome 19

A single vitrified-warmed embryo was transferred in an artificial endometrial priming cycle after priming of the endometrium with oral estradiol valerate (2 mg administered three times a day) (Progynova; Bayer Schering Pharma). When an endometrial thickness of >7 mm was reached, luteal phase support was started with the use of intravaginal micronized progesterone tablets (200 mg three times a day; Utrogestan; Biopas Laboratories). Single embryo transfer was scheduled 5 days later. Embryo transfer (ET) was performed under ultrasound guidance with the use of a soft catheter.

Twelve days after ET, a β-hCG blood test was performed confirming a positive biochemical pregnancy. Six weeks later after ET, the ultrasound scan confirmed a single gestational sac and a heartbeat.

Due to a technology transition, some euploid aCGH were re-analyzed by NGS. Briefly, the same amplified sample was processed following the protocol of Veriseq PGD-AS (Illumina, US). NGS run was performed in a MiSeq. Copy number ratios were analyzed using BlueFuse Multi analysis software (BlueGnome). However, NGS re-analysis indicated that the transferred embryo considered as a euploid for aCGH was mosaic for a monosomy in chromosome 13 and 21; mosaicism was 24 and 34% for each chromosome, respectively (Fig. 2).

At 12 weeks of pregnancy, a 4D genetic ultrasound scan was performed to evaluate the nuchal translucency or other abnormal structures in the fetus due to chromosomal abnormalities. Results showed a normal value of nuchal translucency, and no abnormal structures were found.

At 17 weeks of pregnancy, a non-invasive prenatal test (Verifi ®) which analyzes cell-free fetal DNA circulating in maternal blood for chromosome 9, 13, 16, 18, and 21, and sex chromosome was performed. Results from this test confirmed a euploid fetus development.

Finally, a healthy baby boy was born at 38 weeks; its weight was 4480 g, head circumference 36 cm, and total length of 51 cm. Additionally, NGS test was performed in buccal cells to confirm the existence of mosaic cells. This resulted in a normal NGS profile (Fig. 3).

Fig. 3.

An NGS normal profile obtained from buccal cells

Discussion

In vivo, oocytes are arrested at GV stage because of different regulatory factors and signaling pathway which keep the oocyte immature [24, 25]. However, during an IVM treatment, immature oocytes are retrieved from the follicle environment allowing the spontaneous meiotic resumption [2, 26].

Conventional IVF necessarily requires controlled ovarian hyperstimulation; patients who suffer from PCOS are at risk of development of OHSS, whereas in IVM cycles, these risks decrease [27]. Despite IVM offers certain benefits, this treatment has not been widely used at fertility centers because of the large variation in minimal-stimulation protocols. Some studies have reported the use of hCG trigger prior oocyte retrieval to obtain mature and immature oocytes [28, 29]. However, other clinicians indicate no or low doses of gonadotrophins instead of hCG trigger [30, 31] resulting in the recruitment of a homogeneous cohort of oocytes at GV stage. For this case report, hCG triggering was not administrated.

IVM of human oocytes is a promising treatment for selected patients [32] and should be considered as an alternative for fertility preservation. However, more prospective studies are needed to provide strategies to optimize the clinical outcomes. The extension of the embryo culture at blastocyst stage permits a better embryo selection process [33], because of their post-embryonic genome activation and better development potential [34]. Likewise, numerous studies had indicated higher implantation rates and ongoing pregnancy following frozen-thawed embryo transfer instead of fresh cycles [34–36], this strategy increases the synchronization between embryos and the endometrium. Additionally, embryo cryopreservation prevents OHSS and allows a time frame to perform genetic analyses [20].

Several IVM reports had shown low clinical efficiency, not only for the reduced oocyte quality, but also for insufficient endometrial receptivity. In IVM cycles, embryo cryopreservation had increased implantation and pregnancy rates [31, 37–39]. The majority of the IVM cycles reported had a predilection for cleavage-stage embryo transfer on day 3 [4]. Even though, there is increasing evidence of improving clinical outcomes of IVM cycles when two modifications were applied. First, the reduction of time oocyte culture from 40 to 48 to 30 h, and second, the extension of embryo culture till blastocyst stage [9, 30]. In this report, these two modifications were followed, together with the addition of PGD-AS analysis where the incidence of aneuploidy after IVM cycles has been reported to be similar to conventional IVF in arrested [40] and good quality embryos [41].

Embryo morphology is still the standard method for embryo selection. However, it has been reported that good quality morphology and euploidy are not well correlated [42]. aCGH analysis to perform PGD-AS represents a good tool for embryo selection. Nevertheless, aCGH is not able to discriminate mosaic embryos. On the contrary, NGS detects partial or segmental aneuploidies improving the analysis resolution [43].

Embryo mosaicism is a frequent event in pre-implantational embryo development where the majority of aneuploidies detected are coming from mitotic error [44, 45]. Mosaicism has been observed at all developmental stages of human embryos [46], occurring in 15–90% of cleavage stage [47] and 30–40% of blastocysts in human embryos [47, 48]. In prenatal stages, the incidence of mosaicism is reduced up to 1–2% [47]. This reduction could be explained by the self-repairing mechanisms throughout the development process and thus, making mosaicism undetectable at the time of NIPT test [49, 50].

Several studies have focused on the implantation potential of mosaic embryos, based on the recent evidence reported about pregnancies and healthy live births after the transfer of mosaic blastocysts [51, 52]. Implantation and ongoing pregnancy rates of mosaic embryos are less in comparison between euploid embryos (15.4 and 30.1% in mosaic embryos vs. 46.2 and 55.8% in euploid embryos, respectively) [53]. Likewise, mosaic embryos could be considered for embryo transfer according to the chromosome involved and type of chromosome abnormality. Genetic guidelines have suggested that mosaic embryos with monosomies should be preferentially transferred over trisomies [54, 55], mainly due to some trisomies that are compatible with pregnancy, and children could develop severe congenital abnormalities [56].

This report, based on the several studies conducted through all developmental stages, increases the available data of safety transfer of mosaic embryos, and it is in agreement with genetic guidelines where mosaic embryos could be considered for embryo transfer when monosomies are detected [55].

Finally, the current report describes an IVM treatment which resulted in a healthy baby born. It is important to highlight that this case report involves multiple analysis procedures at different time of embryo development which should be interpreted with caution. Nevertheless, further studies should be conducted to understand the potential development of a mosaic embryo in ART.