{"paper_id":"85e3d512-2703-4cb1-a96a-93305d178b2b","body_text":"Despite assisted reproduction techniques (ART) have advanced significantly since the\nfirst in vitro fertilization baby was born, many issues related to the health of ART\nbabies have been raised. Reports comparing babies born from natural pregnancies and\nthose born from assisted reproduction showed correlations between ART pregnancies\nand worse perinatal outcomes, for instance, preterm birth, low birth weight, small\nfor gestational age, and perinatal mortality ( Fauser\n et al ., 2014 ;  Helmerhorst  et al ., 2004 ;  Ombelet  et al ., 2016 ;  Pandey  et al. , 2012 ;  Sunkara  et al ., 2015 ).\nUndoubtedly, intrinsic parental characteristics must influence baby birth and health;\nhowever, they are insufficient to explain the differences between babies born from\nnatural or assisted pregnancies, considering that, comparing sons born to the same\nmother, the ART singleton baby tend to have more perinatal complications than\nnon-ART sibling ( Hayashi  et al .,\n2012 ;  Henningsen  et al .,\n2011 ;  Kapiteijn  et al .,\n2006 ;  Pandey  et al .,\n2012 ;  Pinborg  et al .,\n2013 ). Moreover, the majority of ART perinatal complications have been\ncommonly attributed to the higher rate of multiple births; nevertheless, singleton\npregnancies from assisted reproduction also had significantly worse perinatal\noutcomes than natural singleton ones ( Grady\n et al ., 2012 ;  Helmerhorst  et al ., 2004 ;  Pandey  et al ., 2012 ;  Qin  et al ., 2016 ;  Sullivan-Pyke  et al ., 2017 ). It is plausible\nthat ART perinatal outcomes depend on a complex combination of parental\nparticularities and aspects of the treatment itself ( Henningsen  et al ., 2011 ;  Nelson & Lawlor, 2011 ;  Olivennes  et al ., 2002 ;  Palomba  et al ., 2016 ;  Pandey  et al ., 2012 ;  Pinborg  et al ., 2013 ).\nControlled ovarian stimulation (COS) protocols are pointed as potential influencers\nof perinatal outcomes ( Hayashi  et\nal ., 2012 ;  Kapiteijn\n et al ., 2006 ). In fact, some studies reported that\nthere was increased number of preterm births and low birth weight babies born to\nhyper-responder mothers ( Kalra  et\nal ., 2011 ;  Sunkara\n et al ., 2015 ), and that adverse perinatal outcomes\nmay be associated with suboptimal endometrial development due to COS ( Chung  et al ., 2006 ). However,\nthe direct correlation between COS and perinatal outcomes is still characterized by\ncontradictions: when the effects of COS were adjusted for biological and social\nconfounders, such as mother's weight and height, duration of infertility, ethnicity,\nand level of education, it no longer influenced birth weight and other neonatal\ncharacteristics ( Griesinger  et al .,\n2008 ;  Pelinck  et al .,\n2010 ;  Sunkara  et al .,\n2016 ).\nIn addition to the number of retrieved oocytes, the oocyte quality might have a role\nin baby development ( Balaban & Urman,\n2006 ;  Hattori  et al .,\n2014 ;  Mateizel  et al .,\n2013 ;  Rienzi  et al .,\n2011 ;  Shaw-Jackson  et\nal ., 2014 ). Embryo quality and embryo transference stage can\nalso influence perinatal outcomes, although direct correlations are not evident\n( Glujovsky  et al ., 2016 ;\n Dar  et al ., 2013 ;  Fernando  et al ., 2012 ;  Källén  et al .,\n2010 ;  Kalra  et al .,\n2012 ;  Martin  et al .,\n2012 ;  Nakagawa  et al .,\n2016 ;  Oron  et al .,\n2014 ;  Schwärzler  et\nal ., 2004 ).\nThe determination of which aspects of ART pose greater risks of perinatal\ncomplications and how these risks can be minimized is extremely important for\nhealthy baby delivery. Therefore, the goal for the present study was to determine\nwhich cycle characteristics, for instance, ovarian stimulation, laboratorial, and\nclinical outcomes, could be correlated with the perinatal outcomes number of\ngestational weeks to live birth (GW), baby weight at birth (BW), and baby length at\nbirth (BL).\n\nThis cohort study included data obtained from 402 babies born to 307 patients\nundergoing their first controlled ovarian stimulation (COS) followed by\nintracytoplasmic sperm injection (ICSI) cycles and fresh transfer of embryos on\ndays three or five of development. Cycles were performed in a brazilian private\nuniversity-affiliated IVF center between January/2014 and December/2015. Couples\nundergoing ICSI with vitrified/thawed or donated oocytes, surgical sperm\nretrieval, vitrified/thawed embryo transfer, donated embryos, or preimplantation\ngenetic diagnosis/screening were excluded from the analysis.\nThe effects of (i) the total FSH dose administered, (ii) the estradiol peak at\nhCG day, (iii) the number of follicles, (iv) the number of retrieved oocytes,\n(v) the number of mature oocytes, (vi) the fertilization rate, (vii) the number\nof embryos obtained, (viii) the high-quality embryos rate at day two and three,\n(ix) the blastocyst rate, (x) the transference stage (cleavage or blastocyst),\n(xi) the endometrial thickness, and (xii) the implantation rate (gestational\nsacs/ embryos transferred) on the number of GW, BW, and BL were evaluated.\nIn a subsequent analysis, cycles were subdivided according to the American\nAcademy of Pediatrics Intrauterine Growth Curves ( Olsen  et al ., 2010 ). Combined GW, BW and\nbaby sex were used for the classification of Appropriate for gestational age\n(AGA), if the baby weight was between 10-90 th  percentile of the curve\n(n=256), or Small for gestational age (SGA), if the weight was bellow the\n10 th  percentile (n=146).\nWritten informed consent, in which patients agreed to share the outcomes of their\ncycles for research purposes, was obtained, and the local institutional review\nboard approved the study (protocol 410/2012).\nControlled ovarian stimulation was achieved using a daily dose of recombinant FSH\n(r-FSH, Gonal-F ® , Merck KGaA, Darmstadt, Germany). Pituitary\nblockage was performed using a GnRH antagonist (GnRHa,\nCetrotide ® ; Merck KGaA, Darmstadt, Germany). Ovulation was\ntriggered with recombinant human chorionic gonadotrophin (hCG,\nOvidrel ™ , Merck KGaA, Geneva, Switzerland). Oocyte\nretrieval was performed 35 hours later through transvaginal ultrasound ovum\npick-up.\nRetrieved oocytes were maintained in culture medium (Global ® \nfor Fertilization, LifeGlobal, Connecticut, USA) supplemented with 10% protein\nsupplement (LGPS, LifeGlobal, Connecticut, USA) and covered with paraffin oil\n(Paraffin Oil P.G., LifeGlobal, Connecticut, USA) for two to three hours before\nthe removal of the cumulus cells. The surrounding cumulus cells were removed\nafter exposure to a HEPES-buffered medium containing hyaluronidase (80IU/mL,\nLifeGlobal, Connecticut, USA). The remaining cumulus cells were mechanically\nremoved by gently pipetting them with a hand-drawn Pasteur pipette (Humagen\nFertility Diagnostics, Charlottesville, USA). Oocyte morphology was assessed\nusing an inverted Nikon Diaphot microscope (Eclipse TE 300; Nikon, Tokyo, Japan)\nwith a Hoffmann modulation contrast system under 400x magnification, just before\nsperm injection (5 hours after retrieval). Oocytes that had released the first\npolar body were considered mature and were used for ICSI.\nIntracytoplasmic sperm injection was performed in a micro-injection dish prepared\nwith 4-µL droplets of buffered medium (Global ®  w/HEPES,\nLifeGlobal, Connecticut, USA) and covered with paraffin oil on the heated stage\nof an inverted microscope (37.0±0.5ºC). Sperm selection was performed at\n400x magnification. Approximately 16 hours after ICSI, the presence of two\npronuclei and the extrusion of the second polar body confirmed fertilization.\nEmbryos were maintained in a 50-µL drop of culture medium\n(Global ® , LifeGlobal, Connecticut, USA) supplemented with\n10% protein supplement and covered with paraffin oil in a humidified atmosphere\nunder 6% CO 2  at 37ºC for three to five days.\nEmbryos were morphologically evaluated on days two, three, and five using an\ninverted Nikon Diaphot microscope with a Hoffmann modulation contrast system\nunder 400X magnification.\nHigh-quality cleavage-stage embryos were defined as those with all of the\nfollowing characteristics (Alpha Scientists in Reproductive Medicine and ESHRE\nSpecial Interest Group of Embryology, 2011): 3−5 cells on day 2 or 8−10 cells on\nday 3, <15% fragmentation, symmetric blastomeres, the absence of\nmultinucleation, colorless cytoplasm with moderate granulation and no\ninclusions, the absence of perivitelline space granularity and the absence of\nzona pellucida (ZP) dimorphisms. The blastocyst rate was defined as the number\nof embryos that reached blastocyst stage on day five (only the full, expanded,\nhatching, and hatched blastocyst were considered) by the number of embryos in\nculture on day three of development.\nEmbryo transfer was performed on the third or fifth day of development using a\nsoft catheter with transabdominal ultrasound guidance. One to three embryos were\ntransferred per patient, depending on embryo quality and maternal age.\nA pregnancy test (serum β-hCG) was performed 12 days after embryo\ntransfer. All women with a positive test had a transvaginal ultrasound scan\nafter two weeks. Clinical pregnancy was diagnosed when the fetal heartbeat was\ndetected. After childbirth, the GW, BW, BL, baby sex, and presence of\nmalformations at birth were recorded by the patient's gynecologist.\nStatistical analysis was performed using SPSS 21 Software (IBM, New York, USA).\nThe effects of response to COS (FSH dose, Estradiol peak at hCG day, number of\nfollicles, number of retrieved oocytes and number of mature oocytes),\nlaboratorial (fertilization rate, number of obtained embryos, high-quality\nembryo rate at day two and three, blastocyst rate and transference stage), and\nclinical (endometrial thickness and implantation rate) outcomes on the GW, BW,\nand BL were evaluated using linear regression models adjusted for maternal age\nand body mass index (BMI), number of transferred embryos, number of gestational\nsacs and number of born infants. The results are expressed as β (linear\nregression coefficient), 95% confidence interval (CI 95%), and  p\nvalue . The α adopted was 5%.\nIn a subsequent analysis, in which cycles were subdivided AGA and SGA groups and\nthe effects of COS, laboratorial and clinical outcomes were evaluated by general\nlinear models adjusted for maternal age and BMI, number of transferred embryos,\nnumber of gestational sacs and number of born infants. The results are expressed\nas Mean±SD and  p value . The α adopted was 5%.\n\nThe patient demographics, cycle characteristics, and perinatal outcomes are described\nin  Table 1 .\nDescriptive statistics of patients' demographics, cycle characteristics, and\nperinatal outcomes\nPCOS= Polycystic ovary syndrome, COS= Controlled ovarian stimulation,\nFSH= Follicle-stimulating hormone\nThe number of follicles (β=-0.069  p =0.018) and retrieved\noocytes (β=-0.087  p =0.049) were negatively correlated with\nBL ( Table 2 ). The endometrial thickness was\npositively correlated with the number of GW (β=0.198\n p =0.003) and BW (β=28.351  p =0.044). On\naverage, a 1-mm increase in endometrial thickness could prolong pregnancy by 1.4\ndays and increase baby weight by 28 g. No correlation between the perinatal outcomes\nand any other evaluated variable was noted.\nLinear regression predictors for the perinatal outcomes gestational weeks to\nlive birth (GW), baby birth weight (BW) and baby birth length (BL)\nFSH= Follicle-stimulating hormone, β= Regression coefficient, CI:\n95% confidence interval for β. Data was adjusted for maternal age\nand BMI, number of transferred embryos, number of gestational sacs and\nnumber of born infants.\nWhen each baby was classified into AGA and SGA groups, it was observed significantly\nhigher estradiol level at hCG day in SGA babies (3897.01±550.35) compared to\nAGA group (2324.78±101.86,  p =0.006) ( Table 3 ). It was also observed a higher number of retrieved\noocytes in the SGA group (16.70±1.78  vs.  12.92±0.42,\n p =0.042). The endometrial thickness was significantly lower in\nthe SGA group (10.27±0.23  vs.  AGA: 11.68±0.17,\n p =0.029). No other differences were observed between AGA and\nSGA groups.\nCOS, laboratorial and clinical outcomes of Small for gestational age (SGA) or\nAppropriate for gestational age (AGA) infants\nFSH= Follicle-stimulating hormone, SD= standard deviation. Data was\nadjusted for maternal age and BMI, number of transferred embryos, number\nof gestational sacs and number of born infants.\n\nChildren conceived by ART represent a substantial proportion of the population,\nnevertheless, there is still increasing evidence that they have a higher risk of\nperinatal complications, and the mechanisms behind this have not been well\nelucidated ( Pandey  et al .,\n2012 ;  Pinborg  et al .,\n2013 ). The present study showed that specific COS and clinical outcomes\naffect GW, BW and BL.\nThe total amount of FSH administered and the estradiol level at hCG day had no\ninfluence when perinatal outcomes were singly observed, which corroborated previous\nobservations ( Griesinger  et al .,\n2008 ;  Sunkara  et al .,\n2015 ). However, when data was classified according to intrauterine growth\ncurve parameters, it was observed that SGA babies came from cycles with\nsignificantly higher estradiol level, indicating that response to COS influences\nintrauterine growth. Elevated estradiol levels have been correlated to impaired\nembryo implantation potential ( Valbuena  et\nal ., 2001 ), decrease in the duration of the endometrial\nreceptivity window ( Ma  et al .,\n2003 ) and lower pregnancy rates ( Mitwally\n et al ., 2006 ). More recently, estradiol levels were\nalso correlated to poorer obstetric and perinatal outcomes ( Pereira  et al ., 2017 ;  Royster  et al ., 2016 ;  Sokalska  et al ., 2017 ). In fact, it has been\nestablished that estradiol level >3450 pg/ml on the day of hCG administration is\nassociated with greater odds of preeclampsia and delivery of an SGA infant ( Imudia  et al ., 2012 ). The\nlatter is in accordance to what has been reported by this study.\nOur data also showed that BL was negatively influenced by the response to ovarian\nstimulation, such as numbers of follicles and retrieved oocytes. The relation\nbetween COS and BL has been observed mainly in multiple pregnancies studies ( Helmerhorst  et al ., 2004 ;\n Olivennes  et al ., 1996 ).\nMoreover, we also observed a higher number of retrieved oocytes in the SGA group,\nwhich indicates that higher response to COS can be prejudicial to proper embryo\ndevelopment, even when results were adjusted for maternal and cycles\ncharacteristics. This observation corroborates reports that associated higher number\nof oocytes retrieved with increased incidence of low birth weight ( Baker  et al ., 2015 ;  Kalra  et al ., 2011 ;  Sunkara  et al ., 2015 ).\nOur evidence demonstrated that endometrial thickness could influence not only embryo\nimplantation but also perinatal outcomes. On average, a 1-mm increase in endometrial\nthickness could prolong pregnancy by 1.4 days and increase baby weight by 28 g. The\nendometrial thickness was also significantly lower in SGA babies, which supports the\nimportance of this measure before embryo transfer. For the best of our knowledge,\ncorrelations between perinatal outcomes and endometrial thickness at hCG day are\nscarce in the literature and the results obtained emphasizes the need for a more\nphysiologically implantation environment in ART to facilitate the birth of healthier\nbabies.\nThe strength of this study is that the analyses were multiple adjusted to withdraw\nmultiple pregnancies vies, considering the number of transferred embryos, the number\nof gestational sacs and the number of born infants as confounder variables. Even\nthough the number of babies analyzed was limited and maternal medical history and\nsocial habits were lacking, this is the first Brazilian report of the relation\nbetween perinatal outcomes and ICSI characteristics, and it should encourage others\nto report their own outcomes.\nIn conclusion, our findings suggest that higher ovarian response to stimulation and\nsuboptimal endometrial development are associated with adverse perinatal outcomes in\nICSI cycles.","source_license":"CC-BY-4.0","license_restricted":false}