Nicolietal.JournalofOvarianResearch2013,6:1 http://www.ovarianresearch.com/content/6/1/1 REVIEW Open Access Pronuclear morphology evaluation in in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) cycles: a retrospective clinical review Alessia Nicoli1*, Francesco Capodanno1, Ilaria Rondini1, Barbara Valli1, Maria Teresa Villani1, Daria Morini1, Leonardo De Pascalis2, Stefano Palomba1 and Giovanni Battista La Sala1 Abstract Background: The assessment of theembryo quality is crucial to maintain an high pregnancy rate and to reduce therisk of multiple pregnancy. The evaluation ofthe pronuclear and nucleolar characteristics ofhumanzygote have been proposedas an indicatorof embryo development and chromosomal complement. The aim of thecurrent study was to assess therole ofpronuclear morphology evaluation invitro fertilization (IVF) /intracytoplasmic sperm injection (ICSI)cycles. Methods: Retrospective clinical analysis on 755 non-elective transfersof onlyone embryo(ET). Embryo assessment was performed indays 1 and 2. Clinical and biologicaldata were recorded and analyzed according to embryoand/ or pronuclear morphology. Results: Both pronuclear and embryo morphology were significantly related to clinical pregnancy and live-birth rates. No significant difference inclinical pregnancy and live-birth rates was detectedwhen the pronuclear and embryo morphology assessments were combined. Embryo morphologyand maternal age were the only independent predictors of favorable outcome by logistic regression analysis. Conclusions: Pronuclear evaluation is effective to select thebest zygotes if ETis performed atday 1, whereas it did notimprovethe clinical outcomes when combined withembryomorphology evaluation inday 2. Keywords: Embryo morphology, IVF, ICSI, Pregnancy,Pronuclear morphology, Live-birth Background The study of the pronuclear and nucleolar characteris- The selection of the best embryo(s) to transfer is a cru- tics of human zygote has been proposed as an indicator cial point in in vitro fertilization (IVF)/ intracytoplasmic of embryo development and competence [4-10]. Previ- sperm injection (ICSI) procedures to maintain a high ous data have suggested that zygote-score could be an performance in terms of pregnancy rate and to reduce efficient tool for embryo selection if combined with theriskofmultiple pregnancies. embryo morphology evaluation on days 2 and 3 [10,11]. Severalmethodologieshavebeenproposedandemployed In particular, the morphological characteristics of the for embryo selection, but their clinical efficacy is still human zygote may suggest an increased risk of arrest of extremelydebatedandconsideredsuboptimal[1-3]. the embryo [11]. Moreover, to date, there is not defini- tive consensus about the clinical efficacy of the zygote pronuclear morphology.In fact,otherAuthors suggested that embryo morphology has a better prognostic value *Correspondence:[email protected] 1DepartmentofObstetrics,GynecologyandPediatrics,SterilityCentreP. than zygote-score in embryo selection [12-14], and that Bertocchi,ObstetricsandGynecologyUnit,A.O.S.MariaNuova,IRCCS,Reggio pronuclear scoring system has any advantage to evaluate EmiliaandUniversityofModena,ReggioEmilia,Italy embryoquality[15]. Fulllistofauthorinformationisavailableattheendofthearticle ©2013Nicolietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Nicolietal.JournalofOvarianResearch2013,6:1 Page2of6 http://www.ovarianresearch.com/content/6/1/1 Based on these considerations, the current study was Oocyteinsemination aimed to evaluate if the pronuclear morphology evalu- For the conventional IVF procedures, oocytes were cul- ation improves the clinical outcomes in IVF/ICSI when tured individually and inseminated in microdrops of combinedwith embryomorphology assessment. fresh medium (Cook IVF, Melbourne, Australia) under mineraloil with 100,000activated spermatozoa. For ICSI procedure, after the removal of the cumulus and corona Materials and methods cells, oocytes nuclear maturation assessment was per- Patients formed using an inverted microscope to ensure the in- The current was a non-interventional, retrospective sin- jectionofmetaphaseIIoocytesexclusively[18]. gle centrereview. Institutional Review Board approval was not required Assessmentoffertilization/cleavage since all couples included underwent a routine IVF/ICSI Oocyte fertilization was assessed at 18–20 hours (day 1) program in our Reproductive Unit, and no additional/ from insemination/injection and confirmed by the pres- experimentalinterventionwasperformed. enceof2pronuclei(2PN)andthealignmentofnucleolar AllIVF/ICSIcycles performedbetween April 2008 and precursor bodies (NPB). At same time, the pronuclear November2010were reviewed. morphological score was assessed [4]. The observation All cycles characterized by the transfer of one embryo of 2PN was performed using an inverted microscope were selected and included in the final analysis. Were withHoffmanmodulationcontrastat×400magnification excluded cycles in which with more than one oocyte was (TE2000U,NikonCorp.,Japan).Zygotes with simultan- fertilizedandcleaved. eously juxtaposed and centralized PN, nucleoli of large For any cycle we collected all available clinical and bio- size and orientated, and orientation of polar bodies in logicaldata.Specifically,werecordedthepatients'charac- the longitudinal axis of PN were classified as Z1 and teristics, the controlled ovarian stimulation regimens, the considered as the best zygote morphology [4,5,19]. drugs and protocols used for luteal phase support, the Zygotes having all other configurations were classified characteristics of oocytes retrieved and embryos transfer, as Z2. the pronuclear and embryo morphology. Implantation, ET was performed after 48 hrs (day 2) of embryo cul- pregnancy,abortionandlive-birthrateswerealsonoted. ture. During embryo observation five parameters were classified: cleavage symmetry, blastomere shape and size, cytoplasmic aspect, presence of fragmentation and of Ovarianstimulationandoocyteretrieval blastomere multi/micronucleation.Embryos characterized In all cases the controlled ovarian stimulation was by symmetric cleavage, regular blastomeres sheep and achievedusingindividualizedprotocols of recombinant- size, absence of any cytoplasmic anomalies such as dark follicle stimulating hormone (rFSH; Gonal F, Serono, areas, granulations and vacuole, blastomeres multinuclea- Rome, Italy) in down-regulated cycles. A serum estra- tion (≥ 2 nuclei per blastomere) or micronucleation were diol concentrations <50 pg/mL and an absence of folli- considered as grade I embryos. Grade II embryos pre- cles having a mean diameter higher than 10 mm were sented slightly irregular blastomeres sheep and size, frag- considered as criteria for gonadotropin administration. mentation ≤ 10%, and small cytoplasmic vacuoles. Grade The ovarian response was monitored by use of serum III embryos were characterized by asymmetric cleavage, estradiolassaysandserialultrasonographies. highlyirregularblastomeressheepandsize,fragmentation In presence of at least one follicle with a mean diam- among10-30%,darkcytoplasm, cytoplasmic vacuoles and eter equal or higher than 17 mm was observed, 10,000 multinucleation. Finally, grade IV embryos displayed IU human chorionic gonadotropin (hCG; Gonasi, IBSA, Milan, Italy) or 250 μg recombinant hCG (Ovitrelle, asymmetric cleavage, severe irregular blastomeres sheep and size, fragmentation among 30-50%, dark cytoplasm Serono, Rome, Italy) were intramuscularly administered. with massive presence of vacuoles. ETs of grades I and II Oocyte retrieval was performed 34 to 36 hours after embryos were defined as High Quality Embryo ET (HQE hCG administration by ultrasound-guided transvaginal ET), whereas ETs of grades III and IV embryos as Poor aspiration. QualityEmbryoET(PQEET). Semenpreparation Embryotransferandestablishmentofpregnancy Semen samples were collected by masturbation after 3– All patients received intramuscular (100 mg/day; Pron- 5 days of abstinence. The preparation for conventional togest; IBSA, Milan, Italy) or transvaginal supplemental IVF or ICSI was performed following the World Health progesterone (600 mg/day; Prometrium, Rottapharm, Organizationstandardprotocol[16,17]. Milan, Italy)for 15days. Nicolietal.JournalofOvarianResearch2013,6:1 Page3of6 http://www.ovarianresearch.com/content/6/1/1 In each case, biochemical pregnancy was determined embryo quality, insemination technique and pronuclear 12 days after ET by a positive quantitative serum β-hCG morphologyaspredictors. assay >10 IU. In case of positive pregnancy test, the hor- For all tests, the statistical significance level was set at monal support was continued until 35 days after the ET. P<0.05. Clinical pregnancy was defined as one embryo with Analysis was performed using SPSS version 20 for heart beat revealed by transvaginal ultrasonography at 5 MicrosoftOffice. weeks after ET. Because of only one embryo was trans- ferred in each ET, the clinical pregnancy was equivalent Results totheembryoimplantation. In the study period, 2,212 IVF/ICSI cycles were screened and 755 cycles (755/2,212, 34.2%) were included in the final analysis. Specifically, 293 and 462 cycles of IVF Statisticalanalysis (293/755, 38.8%) and ICSI (462/755, 61.2%) programs The normal distribution of continuous variables was were studied, respectively. One thousand and two hun- evaluated with the use of the Kolmogrov-Smirnov test, dred five cycles (1.205/2.212, 54.4%) were excluded be- and continuous data were expressed as the mean ± cause of more then one embryo was transferred, and standard deviation(SD). 252 cycles (252/2.212, 11.4%) because of ET was not Continuous data were analyzed by Student t test for performed for fertilization failure or cryopreservation of unpaired data. For categorical variables, the Pearson Chi alloocytes/embryos. square test was performed; Fisher’s exact test was used A statistically significant higher clinical pregnancy [17/ for the frequency tables when more than 20% of the 139 (12.2%) vs. 39/616 (6.3%); P = 0.017] and live-birth expected values werelower than five. [13/139 (9.4%) vs. 30/616 (4.9%); P = 0.039] rates were To investigate which of the variables studied could observed in ETs with embryos derived from Z1 zygotes best predict the chances of a clinical pregnancy and live- (Z1 ETs) compared to ETs with embryos derived from birth, a binary logistic regression was performed, using Z2 zygotes (Z2 ETs). Similarly, a statistically significant Table1Clinicaloutcomesanalyzedaccordingtoembryoandpronuclearmorphologies HQEET PQEET Pvalue (n.,%) (n.,%) Totalcycles 450,59.6 305,40.4 Maternalage(years) 36.83±3.88 36.48±4.04 0.234 Clinicalpregnancyrate 44^/450,9.8 11/305,3.6 0.001 Singleton 42/44,95.5* 11/11,100.0 0.471 Implantationrate 44/450,9.8 11/305,3.6 0.001 Abortionrate 9/44,20.5 2/11,18.2 0.866 Live-birthrate 34/450,7.6 9/305,3.0 0.007 Gestationalageatbirth(weeks) 37.8±4.2 39.6±1.3 0.213 Birthweight(gr) 2979.3±798.3 3214.6±391.0 0.399 Z1ET Z2ET (n.,%) (n.,%) Totalcycles 139 616 Maternalage(years) 36.22±3.71 36.79±3.99 0.123 Excellentqualityembryos 105/139,75.5 345/616,56.0 <0.001 Clinicalpregnancyrate 16^/139,12.2 39/616,6.3 0.017 Singleton 16/16,100.0 37/39,94.9* 0.356 Implantationrate 16/139,11.5 39/616,6.3 0.034 Abortionrate 2/16,12.5 9/39,23.1 0.712 Live-birthrate 13/139,9.4 30/616,4.9 0.039 Gestationalageatbirth(weeks) 37.3±5.1 38.5±3.1 0.351 Birthweight(gr) 2959.6±849.7 3058.4±692.2 0.691 ^:oneectopicpregnancy;*:twosetsofmonozygotictwins. Nicolietal.JournalofOvarianResearch2013,6:1 Page4of6 http://www.ovarianresearch.com/content/6/1/1 Table2Clinicalpregnancyandlive-birthrateanalyzedaccordingtopronuclearandembryomorphology Clinicalpregnancyrate Live-birthrate No Yes Total No Yes Total (n.,%) (n.,%) (n.,%) (n.,%) (n.,%) (n.,%) HQEET Z1 90,85.7 15,14.3 105,100 93,88.6 12,11.4 105,100 Z2 316,91.6 29,8.4 345,100 323,93.6 22,6.4 345,100 PQEET Z1 32,94.1 2,5.9 34,100 33,97.1 1,2.9 34,100 Z2 262,96.7 9,3.3 271,100 263,97.0 8,3.0 271,100 Total 700,92.7 55,7.4 755,100 712,94.3 43,5.7 755,100 Thedistributionpronuclearandembryomorphologywassignificantly(P<0.05)differentbetweenpatientswhohadandwhodidnothadaclinicalpregnancy andalive-birth. higher clinical pregnancy [45/450 (10.0%) vs. 11/305 live-birth rates (P = 0.086 and P = 0.997 for HQE-Z1 vs. (3.6%); P = 0.001] and live-birth [34/450 (7.6%) vs. 9/305 HQE-Z2 ETs and for PQE-Z1 vs. PQE-Z2 ETs, respect- (3.0%); P= 0.007] rates were observed inHQE ETs com- ively)(Table2). pared toPQEETs(Table1). Finally, using the binary logistic regression to evaluate InTable2areshowedtheclinicalpregnancyandlive-birth the best predictor of clinical pregnancy and live-birth ratesperETaccordingtoPNandembryomorphology. only the embryo morphology and the maternal age The distribution of PN and embryo morphology was resultedindependent predictors (Table 3). significantly different (P < 0.05) between patients who had and who did not had a clinical pregnancy and/or a Discussion live-birth (Table 2). The HQE-Z1 and PQE-Z2 ETs To date, the evaluation of pronuclear and embryo morph- showed, respectively, the highest and lowest clinical ologiesisacommonpracticeinARTlaboratoriestoselect pregnancy and live-birth rates (Table 2). No significant thebestembryostotransfer.Evenifseveraldataregarding difference between HQE-Z1 and HQE-Z2 ETs and be- the clinical efficacy of pronuclear morphology evaluation tween PQE-Z1 and PQE-Z2 ETs was detected in clinical have published [4-13,17], the current is the first clinical pregnancy(P=0.093andP=0.450forHQE-Z1vs.HQE- study reporting reproductive data of patients submitted to Z2ETsandforPQE-Z1vs.PQE-Z2ETs,respectively)and thetransferofoneembryobecauseofonlyoneoocytewas Table3Regressionanalysisusingmaternalage,embryoquality,inseminationtechniqueandpronuclearmorphology aspredictorsofclinicalpregnancyanddelivery B ES P OR 95%CI Lower Upper Constant Clinicalpregnancy −3.845 0.374 <0.001 0.021 Delivery −4.084 0.417 <0.001 0.017 Maternalage Clinicalpregnancy 1.263 0.290 <0.001 3.535 2.002 6.244 Delivery 1.282 0.326 <0.001 3.604 1.904 6.821 Inseminationtechnique Clinicalpregnancy 0.021 0.304 0.946 1.021 0.562 1.854 Delivery 0.074 0.341 0.828 1.077 0.552 2.099 Pronuclearmorphology Clinicalpregnancy 0.481 0.320 0.132 1.618 0.865 3.029 Delivery 0.468 0.359 0.192 1.597 0.791 3.227 Embryoquality Clinicalpregnancy 1.078 0.353 0.002 2.938 1.470 5.871 Delivery 0.974 0.391 0.013 2.648 1.231 5.698 Modelforclinicalpregnancyprediction:χ2(4)=33.445;P<0.001;R2=0.106. Modelfordeliveryprediction:χ2(4)=25.200;P<0.001;R2=0.093. Nicolietal.JournalofOvarianResearch2013,6:1 Page5of6 http://www.ovarianresearch.com/content/6/1/1 fertilized and/or cleaved. In fact, at our knowledge, only Authordetails Salumets et al. [20] included in their analysis reproductive 1DepartmentofObstetrics,GynecologyandPediatrics,SterilityCentreP. dataofasingleembryotransferred,evenifthiswas“elect- Bertocchi,ObstetricsandGynecologyUnit,A.O.S.MariaNuova,IRCCS,Reggio EmiliaandUniversityofModena,ReggioEmilia,Italy.2Departmentof ive”[20]. Psycology,UniversityofBologna,Bologna,Italy. The analysis of this specific cohort of patients permit- ted to exclude a priori the bias of embryo selection per- Received:21September2012Accepted:15December2012 Published:3January2013 formed by the operator, even if the overall ARTclinical outcomeswere, asexpected, verypoor. References Firstly,ourdatasuggestthatbothpronuclearandembryo 1. 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MontagM,LiebenthronJ,KösterM:Whichmorphologicalscoringsystem isrelevantinhumanembryodevelopment?Placenta2011,32:S252–S256. doi:10.1186/1757-2215-6-1 Citethisarticleas:Nicolietal.:Pronuclearmorphologyevaluationin invitrofertilization(IVF)/intracytoplasmicsperminjection(ICSI)cycles: aretrospectiveclinicalreview.JournalofOvarianResearch20136:1. Submit your next manuscript to BioMed Central and 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 redistribution Submit your manuscript at www.biomedcentral.com/submit