AA Yuzpe

Alina Mahfoudh

C Chen

CM DeUgarte

D Dozortsev

E Shalom-paz

Elena Garcia-Cerrudo

Jeong Hee Moon

JH Moon

L Ming

L Rienzi

M Montag

N Sermondade

R Beck-Fruchter

Sara Henderson

Shauna Reinblatt

T Pehlivan

Weon-Young Son

WM Buckett

World Health Organization (WHO)

ZP Nagy

Journal of Ovarian Research

English

Crossref

Moon et al. Journal of Ovarian Research  (2015) 8:22 DOI 10.1186/s13048-015-0150-6CASE REPORT Open AccessSuccessful live birth after transfer of blastocystand frozen blastocyst from rescue ICSI withapplication of polarized light microscopy forspindle examination on unfertilized eggsJeong Hee Moon*, Sara Henderson, Elena Garcia-Cerrudo, Alina Mahfoudh, Shauna Reinblattand Weon-Young SonAbstractThis article aims to report successful live births after transfer of fresh blastocyst or vitrified/warmed blastocystderived from intracytoplasmic sperm injection (ICSI) on day-1 of unfertilized mature eggs (so-called “rescue ICSI”)with spindle examination using polarized light microscopy. Two couples who had rescue ICSI performed achieved apositive pregnancy result after the transfer of a fresh or vitrified blastocyst. The two pregnancies led to the livebirths of a healthy baby boy of 2.72 kg and baby girl of 3.4 kg, respectively.Keywords: Rescue ICSI, Spindle, Polarized light microscopy (PolScope ™), Blastocyst, VitrificationBackgroundEven in patients with normal parameters on semen ana-lysis, total fertilization failure or low fertilization (<20%)in IVF cycles may still occur. This unexpected resultmay be due to various-factors such as oocyte quality, ab-normal sperm, stimulation protocol and disruption ofsperm-egg interaction. ICSI on day-1 unfertilized matureeggs (so-called “rescue ICSI”) [1,2] has been recom-mended in order to result in healthy newborns [3] des-pite reports of poor quality embryos, higher 3PN ratesand a higher incidence of chromosomal abnormalities[4,5]. Low pregnancy rates may also be due to the asyn-chrony between the developing embryo and the endo-metrium [6,7]. In a recent report, reducing the timeinterval between oocyte retrieval and rescue ICSI candecrease oocyte aging [8] and cryopreservation of em-bryos derived from rescue ICSI can be considered forsynchronization to improve the endometrium [6,7]. Inaddition, it has been reported that spindle examination withpolarized light microscopy can help select unfertilizedoocytes before rescue ICSI in order to increase the 2PN* Correspondence: mjhee@yahoo.co.krDepartment of Obstetrics and Gynaecology, MUHC Reproductive Center,McGill University Health Center (MUHC), McGill University, Montreal, QC,Canada© 2015 Moon et al.; licensee BioMed Central.Commons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.rate after confirming that no sperm penetrated the egg [9].In addition, it can decrease the rate of 3PN by avoidingdouble injection of sperm into unfertilized eggs whichhave sperm positioned inside the oocyte without leadingto activation [9]. This report describes two healthy livebirths after blastocyst transfer in cases of fertilization fail-ure in IVF cycles in where rescue ICSI was performedafter examination of spindle with PolScope™. Althoughrescue ICSI is still controversial, this treatment is worthconsidering in order to avoid cycle cancellation.Case presentationCase 1The couple, both aged 30, presented with a diagnosis ofunexplained infertility after a history of secondary infer-tility. The couple previously had undergone four artifi-cial uterine inseminations (IUI). Ovarian stimulation wascarried out by standard procedures and oocyte retrievalwas performed as previously described [10]. Semen wasanalysed based on standard criterion for IVF for motility,concentration and strict morphology as assessed byWorld Health Organization (WHO) [11]. Semen analysisshowed a normal volume of 2 ml, sperm concentrationof 25 million/ml, and 30% progressive motility. Thesperm parameters after processing were a volume ofThis is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Moon et al. Journal of Ovarian Research  (2015) 8:22 Page 2 of 40.5 ml with a concentration of 20 million/ml, 80% motilityand 4% normal morphology. Thirteen oocytes were re-trieved and were inseminated 3 h later with 100,000progressive motile spermatozoa/ml. Sperm sample wasprocessed through three-layer density gradients (95%-70%-50%) of Pure sperm (Nidacon, Sweden) followed by twowashes in gamete buffer (Cook, Australia). Fertilizationwas checked 18 h after insemination with an invertedmicroscope at 20 X under Hoffman modulation contrast.None of the oocytes showed signs of fertilization (i.e., pres-ence of two pronuclei (2PN)) and had insufficient sperm(less than 5 sperm) bound to zona pellucida [8]. RescueICSI was promptly performed (21 h after oocyte retrieval)after spindle evaluation to eleven unfertilized mature(MII) eggs that had one spindle with one polar body(PB) visualized (Figure 1a) and one unfertilized MIIwith two-spindles (Figure 1b) was excluded. Consentfrom patient was received before injection. Six out ofeleven showed 2PN in the afternoon (~6 h after injec-tion) on same day of injection. All zygotes were cultureduntil day 5 (~116 h) after injection day. One blastocystwas transferred to the patient’s uterus and three blasto-cysts were cryopreserved for future use using vitrifica-tion method. A serum ß-hCG level of 189 IU/L wasdetected 11 days after embryo transfer. A positive fetalheart beat was confirmed at 6 weeks by a sonographyand a healthy boy was delivered vaginally at 38 weeksweighing 2.72 kg.Case 2A 27 year old patient was assessed for IVF-ET due toprimary infertility with PCOS. Ovarian stimulation wascarried out by standard procedures and oocyte retrieval wasperformed as previously described [10]. The patient re-ceived ovidrel and transvaginal ultrasound-guided oocyteretrieval was performed 35 h later. Eight eggs were re-trieved and were inseminated 3 h after oocyte retrievalFigure 1 Unfertilized MII eggs with one-spindle (a) or two-spindle (b)bar; 50 μm).with sperm concentration of 100,000 per ml. Semenanalysis showed a normal volume of 4 ml, sperm con-centration of 30 million/ml, and 30% progressive motility.The sperm parameters after processing were a volume of0.6 ml with a concentration of 40 million/ml, 85% motilityand 4% normal morphology. At 17 h post inseminationthere was no sign of fertilization (2PN) for eight oocytesand all had insufficient sperm bound to the zona pellucida[8]. Eight unfertilized oocytes with the appearance ofone spindle and PB were chosen for rescue ICSI (21 hafter egg retrieval) which resulted in the fertilization ofsix eggs in the afternoon on injection day. Embryoswere cultured in incubator at 37°C with 6% CO2 and 5%O2 until blastocyst stage for 5 days (~116 h). Oneblastocyst was transferred into the female’s uterus andtwo blastocysts were vitrified for future use. Equilibra-tion medium containing 7.5% (v/v) ethylene glycol (EG)and 7.5% (v/v) 1,2- dimethylsulfoxide (DMSO) and vitri-fication medium containing 15% (v/v) EG, 15% (v/v)DMSO and 0.5 M sucrose were used for blastocyst vitri-fication with Cryotop (Kitazato, Japan) vitrification de-vice. Serum ß-hCG level 16 days after egg collectionwas 27 IU/L and unfortunately resulted in an ectopicpregnancy. The patient returned months later forvitrified-warmed embryo transfer. For warming of vitri-fied blastocysts, they were first exposed to basic HEPESmedium containing 1.0, 0.5, 0.25 M sucrose sequentiallyand then were washed twice in basic HEPES medium.One blastocyst was transferred into the patient’s uterusfollowing assisted hatching. In this cycle, the patientwas treated with oral estrace to prepare the endomet-rium, which reached adequate thickness (≥8 mm). Thephase was complemented by administration of vaginalprogesterone. Embryo transfer was performed on day 5of progesterone administration. Estrogen and progester-one were continued until the pregnancy test. A serumß-hCG concentration was determined 11 days afterexamined with Polscope ™ (arrow; PBs, arrow head; spindles,Moon et al. Journal of Ovarian Research  (2015) 8:22 Page 3 of 4embryo transfer. The level of ß-hCG was 1059 IU/L anda positive fetal heart beat was confirmed at 6 weeks bysonography. At 37 weeks, the patient delivered a healthygirl weighing 3.4 kg.DiscussionIt has been reported previously that unfertilized MIIoocytes with only one spindle visualized using PolScope™after IVF may be due to sperm penetration failure whereasMII oocytes with two spindles results from oocyte activa-tion failure (even if sperm entered the oocytes) [9]. In suchcases, performing rescue ICSI as early as possible withoutany delay to verify appearance of later PN formation mayresult in embryos with higher viability [8,9]. Recently, inour center, prompt rescue ICSI (16 ~ 18 h after initialinsemination) for those eggs with one spindle uponvisualization was performed to reduce oocyte aging andavoid the possibility of double insemination. Spindlepresence was observed with the polarization microscopeand rescue ICSI was performed promptly after checkingthe spindle in the same glass bottom dish at 37°C [8].When we examined the spindle of unfertilized eggs justbefore rescue ICSI, there were two other types of spin-dle appearance examined with PolScope™: zero spindle(no visualization of spindle) and mitotic spindle (ourunpublished data). The absence of spindle can be seenduring the time of transition from MI to MII associatedwith low fertilization rate [12,13] or in some unfertilizedeggs which lacked signs of spindle presence in the morningbut showed 2PN that afternoon without rescue ICSI per-formed (in our preliminary data not shown). In addition,the mitotic spindle was seen as one big spindle in the mid-dle of the cytoplasm which is thought to be the mitoticplate for embryo development after having missed assess-ment of 2PN formation but can still produce viable blasto-cysts (data not shown). Therefore, unfertilized eggs withzero spindle or mitotic spindle were also excluded fromrescue ICSI along with those showing two spindles.Fertilization assessment for oocytes that have under-gone rescue ICSI was performed in the afternoon of thesame day as injection because it has been suggested thatpronuclei for 1-day-old human oocytes inseminated afterretrieval can already be seen under the inverted micro-scope as early as 6 h after ICSI [14]. The embryos couldresult in good quality blastocysts after culturing for5 days with the possibility of additional good qualityblastocysts for cryopreservation.It is possible that the disappointing outcomes afterrescue ICSI could be due to the asynchrony between em-bryo development and the endometrial secretory patternas well as oocyte aging [7]. Therefore, cryopreservationof good quality blastocysts for subsequent transfer invitrified-warmed cycles could be a good strategy for over-coming poor outcomes due to asynchrony [6].The main question in cycles with failed fertilization iswhether to perform rescue ICSI or to cancel the cycle.After all, the goal of assisted reproductive technology(ART) is to help infertile couples have a healthy baby, notto simply increase the number of transferable embryos.However, previously reported data [15] demonstrates onlyone out of twenty pregnancies was diagnosed and termi-nated due to trisomy 21 while the other children werefound to be healthy after delivery.It should also be noted that patients have alreadyinvested their time and money for IVF treatment. There-fore, we must also consider the emotional burden thatcycle cancellation would have. At our center, the cost ofIVF treatment is publicly funded therefore eliminatingthe financial burden on couples, however single embryotransfers are the standard. On the other hand, thereis still the emotional disappointment if cycles are can-celled. We therefore have adopted a policy of culturingto blastocyst stage in order to perform elective singleembryo transfer. We suggest that in well-selected pa-tients, rescue ICSI may be considered as an alternativemethod to avoid cycle cancellation by producing viableblastocysts which lead to normal and healthy babies.ConclusionsEarly rescue ICSI after spindle examination with PolScope™could decrease the effects of in vitro aging in 1-day-old-unfertilized oocytes and result in a successful pregnancywith the formation of good quality blastocysts for transferor cryopreservation.ConsentsWritten consent with sign were obtained from patientsfor publication of this case reports.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsJHM performed rescue ICSI after examining spindle with PolScope™,conception and design. SH, EGC and AM helped the revision of article. SRand WYS performed interpretation and revision of the article. All authorsread and approved the final article.Received: 15 December 2014 Accepted: 23 March 2015References1. Nagy ZP, Joris H, Liu J, Staessen C, Devroey P, Van Steirteghem AC.Intracytoplasmic single sperm injection of 1-day-old unfertilized humanoocytes. Hum Reprod. 1993;8:2180–4.2. Yuzpe AA, Liu Z, Fluker MR. Rescue intracytoplasmic sperm injection(ICSI)-salvaging in vitro fertilization (IVF) cycles after total or near-totalfertilization failure. Fertil Steril. 2000;73:1115–9.3. Beck-Fruchter R, Lavee M, Weiss A, Geslevich Y, Shalev E. Rescueintracytoplasmic sperm injection: a systematic review. Fertil Steril.2014;101:690–8.4. Pehlivan T, Rubio C, Ruiz A, Navarro J, Remohi J, Pellicer A, et al. Embryonicchromosomal abnormalities obtained after rescue intracytoplasmic spermMoon et al. Journal of Ovarian Research  (2015) 8:22 Page 4 of 4injection of 1-day-old unfertilized oocytes. J Assist Reprod Genet.2004;21:55–7.5. DeUgarte CM, Li M, Jordan B, Hill D, DeCherney A, Surrey M. Rescueintracytoplasmic sperm injection and preimplantation genetic diagnosis incombination can result in pregnancy. 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Gynecol Endocrinol. 2011;27:993–6.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color ﬁgure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submit

Successful live birth after transfer of blastocyst and frozen blastocyst from rescue ICSI with application of polarized light microscopy for spindle examination on unfertilized eggs

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Successful live birth after transfer of blastocyst and frozen blastocyst from rescue ICSI with application of polarized light microscopy for spindle examination on unfertilized eggs

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