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Table of Contents
CASE REPORT
Year : 2020  |  Volume : 3  |  Issue : 1  |  Page : 42-45

Live birth following the transfer of a euploid blastocyst derived from monopronuclear zygote


1 Reproductive Medicine Unit, Ferticity Fertility Clinics, Delhi, India
2 Department of Reproductive Medicine, Mother and Child Hospital, Delhi, India

Date of Submission07-Jul-2020
Date of Acceptance07-Jul-2020
Date of Web Publication30-Jan-2021

Correspondence Address:
Dr. Surleen Kaur
Ferticity Fertility Clinics, 12, Navjeevan Vihar, Malviya Nagar, Delhi - 110 017
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tofj.tofj_1_20

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  Abstract 


The objective of the present study is to report a case of a live birth following the transfer of a blastocyst derived from monopronuclear zygote that was found to be an euploid by preimplantation genetic testing for aneuploidy (PGT-A). The study was conducted at a tertiary care-assisted reproductive technology center. A 35-year-old female with a secondary infertility, polycystic ovaries, and with the history of recurrent implantation failure underwent an in vitro fertilization cycle followed by a frozen embryo transfer cycle. The main outcome measure was clinical pregnancy and live birth. The patient was considered pregnant after obtaining a βhCG level of 1334 mIU/mL after 14 days following embryo transfer and delivered a healthy baby girl at 30 weeks by cesarean section. In this study, transfer of frozen-thawed monopronuclear-derived blastocyst that was detected euploid with PGT-A, resulted in a healthy live birth. Therefore, we suggest that embryos from monopronuclear zygotes should not be discarded but instead be screened by PGT-A to ascertain the euploid status, especially in the absence of bipronuclear-derived embryos.

Keywords: Blastocyst, fertilization, intracytoplasmic sperm injection, monopronuclear zygote, preimplantation genetic testing for aneuploidy


How to cite this article:
Kaur S, Nandi K, Gupta S, Sehrawat N. Live birth following the transfer of a euploid blastocyst derived from monopronuclear zygote. Onco Fertil J 2020;3:42-5

How to cite this URL:
Kaur S, Nandi K, Gupta S, Sehrawat N. Live birth following the transfer of a euploid blastocyst derived from monopronuclear zygote. Onco Fertil J [serial online] 2020 [cited 2021 Feb 24];3:42-5. Available from: https://www.tofjonline.org/text.asp?2020/3/1/42/308403




  Introduction Top


Assisted reproductive technologies (ARTs) provide an opportunity to infertile couples to have a child. It also enables scientists to monitor human preimplantation embryo development from gametes till blastocyst stage in vitro. After the fusion of gametes through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), normal fertilization is observed after 16–18 h postinsemination. Normal fertilization is assessed by the extrusion of the second polar body (PB) and the presence of two pronuclei (PN) that determines the diploid status of the zygote.[1] Aberrant zygotic behaviors, such as the presence of 1PN or more than 2PN, are considered to be abnormal with aneuploidy risk and are hence excluded from clinical use as per the European Society of Human Reproduction and Embryology guideline group on good practice in IVF laboratories.[1],[2] However, few studies have reported that embryos derived from 1PN may lead to normal diploid embryos.[3],[4],[5],[6],[7] In view of this, several mechanisms have been proposed to explain the diploid nature of embryos derived from monopronuclear (1PN) zygotes including asynchronous PN formation, male and female PN fusion (karyogamy), premature breakdown of pronuclear membrane, and common pronuclear membrane for both PN.[3],[8],[9] Such zygotes contain an equal distribution of paternal and maternal genome and hence need an assessment reconsideration.

Although a very small percentage of 1PN-derived embryos have the potential to develop till the blastocyst stage, they were found to have comparable clinical outcomes with 2PN-derived blastocyst as reported in conventional IVF (cIVF) studies.[4],[5],[6],[10],[11] Moreover, one of the studies has suggested that the blastocyst derived from larger size 1PN has a better developmental potential and is suspected to be diploid.[5] In contrast to cIVF, the clinical outcomes for ICSI-derived 1PN embryos in terms of pregnancy and live birth rates (LBR) were found to be very low (4%).[6] One of the major reasons is that, during cIVF, sperm enters the oolemma and is close to the spindle apparatus in the ovular cytoplasm. This increases the probability of the paternal and maternal chromosomes being in close proximity before nuclear membrane formation and thus resulting in more frequent occurrence of diploid 1PN by cIVF.[6],[12] Furthermore, despite the fact that the presence of Y chromosome confirmed paternal fertilization in 1PN-derived embryos, the majority of these embryos were found to be aneuploid.[11] Therefore, these embryos should be screened by preimplantation genetic testing for aneuploidy (PGT-A) to confirm their euploid status and should be considered for transfers only when there are no normally fertilized embryos left for the patient. The present study reports one such case of a healthy live birth from a single 1PN blastocyst obtained by ICSI, diagnosed euploid with PGT-A, and then transferred in a frozen embryo transfer (FET) cycle.


  Case Report Top


A 35-year-old female presented to our IVF clinic with secondary infertility, polycystic ovaries, and with a history of recurrent implantation failure (RIF). She had taken antituberculosis treatment in 2011 for genitourinary tuberculosis. Strong family history of diabetes was present among parents and grandparents. She had one spontaneous conception in 2015, which was medically terminated at 16 weeks in view of nonimmune hydrops. Karyotype analysis of the aborted fetus was reported normal via Fluorescence in situ hybridization (FISH). In her investigations, tumor necrosis factor-alpha was found to be raised. Antiphospholipid profile, thrombophilia screening, and karyotyping of both the partners were normal. Mild oligoasthenospermia was present, for which the male partner was put on antioxidants. She underwent two fresh and two FET cycles at other clinics after the first conception. In the first IVF cycle, she had a fresh embryo transfer (three day 3, Grade A embryos). In the second IVF cycle, fresh embryo transfer was done at day 5 where three Grade A blastocysts were transferred. Further two cycles of FET were undertaken, and both times three day 3 embryos were transferred. All these transfers resulted into βhCG levels < 10mIU/mL after 14 days following the embryo transfer.

In view of this history of cycles, hysteroscopy was performed first to identify the cause of RIF, and upon examination, the uterine cavity was found to be normal. Because of RIF and advanced age, the patient was suggested an IVF cycle with PGT-A. Dietary supplementation with antioxidants, folic acid, Vitamin D, and ecosprin was started. Ovarian stimulation was done with recombinant FSH (Recagon, Organon India Pvt. Ltd.) and highly purified menotropin (Menopur, Ferring Pharmaceuticals). GnRH antagonist 0.25 mg (Vestova, Ordain Healthcare) was added on day 6. The total duration of stimulation was 9 days. The total gonadotropin dose was 2850 IU. Trigger was given with GnRH agonist (Decapeptyl 0.2 mg, s/c Ferring Pharmaceuticals). On the day of trigger, serum estradiol was 3083 pg/ml and serum progesterone was 2.27 ng/ml. A total of 17 oocytes were retrieved, only ten were mature oocytes, and ICSI was performed on these. Fertilization was assessed 17 h after microinjection. Seven oocytes were normally fertilized (2PN) and one oocyte was found to be 1PN (monopronuclear) with 2PBs. An image of the monopronuclear zygote is shown in [Figure 1]a. Reassessment of fertilization status of oocyte with 1PN after 4 h of the first fertilization check showed complete disappearance of PN. Irrespective of the number of PN, all eight zygotes were cultured in separate 30 μL drops of one-step culture medium (SAGE, Origio, Denmark) and taken to blastocyst stage for genetic screening. On day 5, three blastocysts were formed. One Grade A blastocyst derived from 2PN zygote, one Grade C blastocyst derived from 2PN zygote, and one Grade A blastocyst derived from monopronuclear zygote [Figure 1]b. Out of the three, one Grade C blastocyst that was not of good quality to withstand biopsy and hence, was transferred in fresh cycle, whereas the two blastocysts of Grade A quality were biopsied and cryopreserved. Biopsied samples were sent for PGT-A to the molecular laboratory (Igenomix Laboratory, Spain). Surprisingly, the blastocyst that was derived from 1 PN was reported as euploid, whereas the blastocyst developed from 2PN was identified as low mosaic aneuploid (-5,-16). Once the fresh embryo transfer result was found to be negative, the patient gave consent for the transfer of the only euploid blastocyst left, derived from 1 PN. Because of RIF history, endometrial receptivity array (ERA) was performed in a hormone replacement cycle to identify the exact “window of implantation” (WOI). Estradiol valerate (Progynova, Zydus Cadila) was started from day 2 of the cycle after a baseline transvaginal scan. Progesterone (P) vaginal pessary 400 mg twice a day was added when the endometrial thickness reached 8 mm, and serum progesterone level on the day of the start of progesterone was 0.6 ng/ml. Endometrial biopsy for ERA was taken after 5 days of progesterone administration (P + 5). ERA result showed that the “WOI” was altered, and embryo transfer was advised at 112 ± 3 h.
Figure 1: (a) Image of human zygote with monopronucleus (1 pronuclei, as indicated by arrow) obtained 17 h postintracytoplasmic sperm injection with the presence of the first and second polar body. (b) Blastocyst derived from monopronuclear zygote on day 5 of embryo development (ZP: zona pellucida, PVS: perivitelline space, PB: polar Body, TE: trophectoderm, ICM: inner cell mass)

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For the subsequent FET, endometrial preparation was done in the exact way as for ERA testing. Progesterone supplementation was initiated on day 12 of cycle when endometrial thickness was 8.1 mm, trilaminar pattern, and serum progesterone was <0.1 ng/ml. At P + 112 h, a hatching blastocyst was transferred to the patient. The patient was considered pregnant after obtaining a βhCG level of 1334 mIU/mL after 14 days following embryo transfer. Level 1 and 2 scans done at 12- and 20-week gestation did not reveal any abnormality. Noninvasive prenatal screening (24c) was done at 13 weeks and did not show any chromosomal abnormality. The patient delivered a healthy baby girl at 30-week gestation by cesarean section. Early intervention was due to premature rupture of membranes and pregnancy-induced hypertension.


  Discussion Top


The aim of the present study is to report a case of healthy live birth from a 1PN-derived blastocyst which was genetically identified as euploid by PGT-A and hence transferred in a frozen-thawed transfer cycle.

There are various methods of detecting euploidy and parental ploidy in 1PN-derived embryos. The most noninvasive and visually efficient method to verify asynchronous appearance of PN is a reassessment of fertilization after 4–6 h of the first fertilization check. In this regard, time-lapse microscopy helps in a noninvasive manner to detect the pronuclear breakdown (PNBD) in diploid 1PN embryos. It allows the development of software-aided PN area and diameter cutoff (≥710 μm or ≥31 μm, respectively) for blastocyst developmental potential of 1PN-derived embryos using receiver operating characteristic curve analysis.[7] Zygotes surpassing these cutoffs have a greater blastulation potential. In 2009, Liao et al. noted a significantly lower blastocyst formation rate for 1PN embryos than 2PN embryos, underlining the importance of extended in vitro culture in the selection of viable, usable embryos. They also noted a greater diploidy rate in 1PN blastocysts (74.6%) than in 1PN-arrested embryos (31.6%).[10] 1PN-derived embryos with gynogenesis and androgenesis impair chromosomal makeup which causes cell fragmentation and apoptosis, resulting in poor blastocyst potential. Parental ploidy selection is, hence, also aided by extending in vitro culture till day 5/6 in cIVF.[4] Immunocytochemistry also confirmed the role of female-PN, male-PN, and PNBD in IVF and ICSI 1PN-derived embryos. FISH combined with antibody labeling for the presence and absence of lamin phosphorylation and chromosomal structural proteins Me (3) H3K9 showed premature breakdown of nuclear envelope in embryos derived from monopronuclear zygote.[2],[13]

Over time, genetic screening has gained immense importance in determining complete parental genome inheritance in embryos developed from monopronuclear zygotes. Zygotes with 0PN and 1PN are found with a frequency of 11.3%–20% and 1.6%–7.7%, respectively.[6],[14] Genome-wide haplotyping of competent 0PN and 1PN-derived blastocysts by Destouni et al. for PGT for a monogenic disorder rescued 31% cycles to reach the embryo transfer.[15] Identification of these embryos as diploid, biparental, and unaffected for the genetic disorder increased the LBR from 9.52% to 16.7%. Comparative analysis of LBR for 1PN and 2PN embryos by Hondo et al. stated the outcomes for IVF-1PN-derived embryos to be similar to those achieved with 2PN embryos.[6] ICSI-1PN embryos, however, had a poor LBR (4%). Previously, chromosomal analysis using FISH has also shown a significantly higher rate of diploid chromosome constitution in embryos from cIVF-1PN than that in ICSI-1PN.[16] The present case study, however, reports that, despite a low LBR and PR, ICSI-1PN embryos should not be completely ruled out for transfer in cases where no 2PN embryos are present. With a genetic screening, such embryos show optimum potential to result in a healthy live birth in compromised ART cases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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De los Santos MJ, Apter S, Coticchio G, Debrock S, Lundin K, Plancha CE, et al. Revised guidelines for good practice in IVF laboratories (2015). Hum Reprod 2016;31(4):685-6.  Back to cited text no. 1
    
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Bradley CK, Traversa MV, Hobson N, Gee AJ, McArthur SJ. Clinical use of monopronucleated zygotes following blastocyst culture and preimplantation genetic screening, including verification of biparental chromosome inheritance. Reprod Biomed Online 2017;34:567-74.  Back to cited text no. 3
    
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Itoi F, Asano Y, Shimizu M, Honnma H, Murata Y. Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes. J Assist Reprod Genet 2015;32:1401-7.  Back to cited text no. 4
    
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Otsu E, Sato A, Nagaki M, Araki Y, Utsunomiya T. Developmental potential and chromosomal constitution of embryos derived from larger single pronuclei of human zygotes used in in vitro fertilization. Fertil Steril 2004;81:723-4.  Back to cited text no. 5
    
6.
Hondo S, Arichi A, Muramatsu H, Omura N, Ito K, Komine H, et al. Clinical outcomes of transfer of frozen and thawed single blastocysts derived from nonpronuclear and monopronuclear zygotes. Reprod Med Biol 2019;18:278-83.  Back to cited text no. 6
    
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Araki E, Itoi F, Honnma H, Asano Y, Oguri H, Nishikawa K. Correlation between the pronucleus size and the potential for human single pronucleus zygotes to develop into blastocysts: 1PN zygotes with large pronuclei can expect an embryo development to the blastocyst stage that is similar to the development of 2PN zygotes. J Assist Reprod Genet 2018;35:817-23.  Back to cited text no. 7
    
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Kai Y, Iwata K, Iba Y, Mio Y. Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number. J Assist Reprod Genet 2015;32:1589-95.  Back to cited text no. 9
    
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Liao H, Zhang S, Cheng D, Ouyang Q, Lin G, Gu Y, et al. Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes. J Assist Reprod Genet 2009;26:583-9.  Back to cited text no. 10
    
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Si J, Zhu X, Lyu Q, Kuang Y. Obstetrical and neonatal outcomes after transfer of cleavage-stage and blastocyst-stage embryos derived from monopronuclear zygotes: A retrospective cohort study. Fertil Steril 2019;112:527-33.  Back to cited text no. 11
    
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Noyes N, Fino ME, Krey L, McCaffrey C, Adler A, Grifo J. Embryo biopsy: The fate of abnormal pronuclear embryos. Reprod Biomed Online 2008;17:782-8.  Back to cited text no. 12
    
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van der Heijden GW, van den Berg IM, Baart EB, Derijck AA, Martini E, de Boer P. Parental origin of chromatin in human monopronuclear zygotes revealed by asymmetric histone methylation patterns, differs between IVF and ICSI. Mol Reprod Dev 2009;76:101-8.  Back to cited text no. 13
    
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Liu J, Wang XL, Zhang X, Shen CY, Zhang Z. Live births resulting from 0PN-derived embryos in conventional IVF cycles. J Assist Reprod Genet 2016;33:373-8.  Back to cited text no. 14
    
15.
Destouni A, Dimitriadou E, Masset H, Debrock S, Melotte C, Van Den Bogaert K, et al. Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M. Hum Reprod 2018;33:2302-11.  Back to cited text no. 15
    
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Yan J, Li Y, Shi Y, Feng HL, Gao S, Chen ZJ. Assessment of sex chromosomes of human embryos arising from monopronucleus zygotes in in vitro fertilization and intracytoplasmic sperm injection cycles of Chinese women. Gynecol Obstet Invest 2010;69:20-3.  Back to cited text no. 16
    


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