{"paper_id":"fecb514c-914d-41da-94e4-cba4c6224834","body_text":"R E S E A R C H Open Access\nSubendometrial resistence and pulsatility\nindex assessment of endometrial\nreceptivity in assisted reproductive\ntechnology cycles\nR. Silva Martins 1,2*, A. Helio Oliani 1, D. Vaz Oliani 1 and J. Martinez de Oliveira 1,2\nAbstract\nObjective: To evaluate Subendometrial and Uterine artery resistance and pulsatility index continuous analysis as a\npredictor of Endometrial receptivity in Assisted Reproductive Technology (ART) Cycles.\nDesign: Serial 2D transvaginal coloured power doppler ultrasound performed in women on ART cycle to evaluate a\npattern that better predicts implantation rates. One hundred sixty-nine subjects on a prospective case control study\nwere assessed. Uterine artery and Subendometrial resistance and pulsatility index was performed to all subjects at\nbaseline (prior to ovarian controlled stimulation), at day 6, 8 and 10 of controlled ovarian stimulation, at trigger day\nand at embryo transfer day. Also the ratio of fluxometric parameters between Subendometrial blood flow and\nuterine artery was measured.\nResults: No statistical difference was noted between two groups in terms of demographics and ART procedures\nand scores. Uterine artery resistance and pulsatility index showed statistical difference between the two groups\n(implantation versus non-implantation group). Also statistical significance was obtained between two groups in\nterms of Subendometrial vascularization. Ratio between Subendometrial and Uterine artery showed lower values of\nfluxometric parameters in all range for the Subendometrial territory.\nConclusions: Serial Subendometrial and Uterine artery fluxometry may be a useful tool for clinicians in predicting\nendometrial receptivity enhancing elective embryo transfers in the same ART cycle.\nKeywords: Endometrial receptivity, Assisted reproductive technology, Subendometrial blood flow, Uterine artery\nfluxometry, Embryo implantation\nIntroduction\nHuman implantation is a complex process requiring syn-\nchrony between a healthy embryo and a functionally\ncompetent or receptive endometrium [ 1]. Since the\nintroduction of assisted conception, many techniques\nhave been developed to further improve ovarian stimula-\ntion, oocyte retrieval, and embryo culture [ 2]. However\nthere has always been a lack in understanding the endo-\nmetrial characteristics compatible with a successful\npregnancy. To prepare for pregnancy, the endometrial\nlining in the uterus thickens and becomes receptive to\nimplantation of a fertilized egg. This happens in re-\nsponse to hormone secretion, with oestrogen and pro-\ngesterone being the primary hormones that are released\nto ensure the endometrial lining is receptive to preg-\nnancy [ 3]. Diagnosis of endometrial receptivity (ER) has\nposed a challenge and so far, most available tests have\nbeen subjective and lack accuracy and a predictive value.\nMicroarray technology has allowed identification of the\ntranscriptomic signature of the window of receptivity -\nwindow of implantation (WOI). This technology has led\nto the development of a molecular diagnostic tool, the\nER array (ERA) for diagnosis of ER [ 4]. The ERA is a\n© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0\nInternational License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and\nreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to\nthe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver\n(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\n* Correspondence: renato.alessandre@gmail.com\n1Centro Hospitalar Universitário Cova da Beira EPE, Quinta do Alvito, 6200\n503 Covilha, Portugal\n2Centro Investigação Ciências da Saúde – Faculdade Ciências da Saúde,\nUniversidade da Beira Interior, Alameda Infante D, Henrique, 6200 506\nCovilha, Portugal\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 \nhttps://doi.org/10.1186/s12958-019-0507-6\n\ntissue test, which evaluates the receptivity of the endo-\nmetrial lining to determine the window of implantation.\nIt is performed based on the assumed WOI for a woman\nduring a natural cycle or an HRT (hormone replacement\ntherapy) cycle [ 5]. The test consists of an endometrial bi-\nopsy to determine the optimal timing for implantation\nin a round of Assisted Reproductive Technology (ART)\ncycle [ 6]. Nevertheless, ERA requires an invasive proced-\nure, it has an associated cost, and in women with irregu-\nlar cycles may not prove to be cost-efficient. Ultrasound\nis a non-invasive technique that can assess changes in\nthe endometrium during stimulated cycles. The use of\nhigh-resolution transvaginal probes made possible follow\nup throughout the cycle of endometrium changes [ 7].\nUterine receptivity is regulated by a number of factors\nincluding uterine perfusion, and better yet endometrial\nperfusion. Differences between infertile and fertile\nwomen uterine perfusion have been reported. It has\nbeen suggested that impaired uterine and endometrial\nperfusion may be the cause of failure. In ART cycles\nblood flow resistance in uterine artery and in the endo-\nmetrial territory has been reported to be a predictive in-\ndicator of implantation. However using this method is\nstill controversial in clinical applications, and many stud-\nies reported a small sample size or a single one moment\nobservation. From a clinical point of view some objective\nparameters must be obtained in order to ascertain the\nlikelihood of an on going pregnancy in ART cycles, pref-\nerably in a non-invasive and cost efficient way [ 8]. Many\nstudies have been conducted on the hemodynamic\nchanges of utero-ovarian arteries during ART cycles. For\noptimizing the results of ART it is critical to decide the\nbest timing for embryo transfer. With the introduction\nof high-resolution transvaginal probes, the non-invasive,\naccessible transvaginal sonography made it particularly\nsuitable for serial follow up throughout the stimulated\ncycle. The aim of this prospective study is to further\nevaluate the capability of serial measurement of suben-\ndometrial fluxometry dopppler flow as a non-invasive\nprocedure to determine endometrial receptivity [ 9–11].\nMaterial and methods\nProspective case control study of 169 women in ART cy-\ncles. All data collected and informed written consent\nwas obtain according to the Ethics Committee of our\nInstitution.\nOnly subjects with viable good grade embryos for\ntransfer (double embryo transfer on day 3 of develop-\nment) were selected. All subjects have been in a short\nprotocol regimen with antagonist for ovarian stimulation\nusing gonadotropins. All used recombinant human\ngonadochorionic hormone (rhCG) for induction of ovu-\nlation 36 h prior to oocyte pick up. Demographics data\nwas collected for all patients and serial ultrasound\nanalysis (uterine artery fluxometry and subendometrial\nblood blow) was performed using the same protocol for\nall participating subjects. Colour Doppler signals are\nmeasured in uterine artery and their ascending branches\nlocated in the outer third of the myometrium. The im-\npedance of blood flow through the uterine arteries may\nbe expressed as the pulsatility index (PI), unit less and\nangle independent, and the resistance index (RI), unit\nless and angle dependent. The PI is measured from the\nflow velocity waveforms as the systolic peak velocity\nminus end-diastolic velocity divided by the mean. It can\nbe classified as low (0.00 –1.99), medium (2.00 –2.99) and\nhigh (over 3.0). The RI is calculated as the ratio of peak\nsystolic flow velocity minus end-diastolic velocity di-\nvided by peak systolic velocity, ranging from 0.0 to 1.0.\nSubendometrial blood flow represents vessels irrigating\nendometrium within 10 mm of the lateral endometrium\nborder [ 12–15]. During ovarian controlled stimulation\nserial ultrasound exams were performed and serum estra-\ndiol levels obtained for all participants. Uterine and suben-\ndometrial resistance and pulsatility index was obtained\nwith 2D sagittal uterine view with power coloured doppler\nin all evaluations (Basal moment – Day 2 or 3 of women\nmenstrual cycle and prior to begin of ovarian controlled\nstimulation; at Day 6, Day 8 and Day 10 after the begin of\novarian controlled stimulation; at Trigger day; and at Em-\nbryo Transfer). Blood flow evaluations were performed in\nthe morning to avoid fluctuation due to circadian rhythm\nof uterine artery blood flow [ 16–18].\nAt Day 12 after embryo transfer, human gonadochor-\nionic sub-unit B serum levels were obtained, and groups\nwere set: positive results (for values over 5 International\nUnits - IU) and negative results (for values under 5 IU).\nAll data collected was analysed between these two set\ngroups and compared.\nAll data was analysed using SPSS (Statistical Package\nfor Social Sciences) version 25.0. Results are presented\nas mean values with standard deviation (SD). Compari-\nsons between means among the study groups were per-\nformed using independent samples t-Test. A Value of\np < 0.05 was considered statistically significant.\nThe authors do not report any conflict of interest.\nThe study protocol has been approved by the Ethics\nCommittee of our Institution (CHCB 22/2017), in ac-\ncordance with the relevant guidelines and regulations.\nResults\nUterine artery resistency and pulsatility index, as well as\nsubendometrial blood flow resistance and pulsatility\nindex was obtained in all 169 cycles using 2D power\ndoppler transvaginal ultrasound in continuous observa-\ntions. Demographics characteristics and ART parameters\nare shown in Table 1. Women were divided into two\ngroups depending on the value of hCG at Day 12 after\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 2 of 7\n\nembryo transfer: 123 in the negative group – non-\nimplantation group (72.8%) and 46 on the positive group\n– implantation group (27.2%). There were no statistical\ndifference between two set groups in terms of demo-\ngraphics and ART parameters.\nUterine artery blood flow showed no statistical differ-\nence between two groups at baseline, both for resistance\nand pulsatility index. Statistical difference between two\ngroups is shown after day 6 of ovarian controlled stimu-\nlation for both parameters in analysis (Table 2 and Fig. 1).\nWe can see that both resistance and pulsatility index in-\ncrease its values slightly until trigger day with hCG. The\nresults are however; always lower for the implantation\ngroup. (trigger day with hCG 0.93 ± 0.10 on the non-\nimplantation group versus 0.88 ± 0.09 for the implant-\nation group with p value of 0.011 in the resistance index\nand 1.48 ± 0.38 versus 1.18 ± 0.27 with p value of 0.001\nfor the pulsatility index). After trigger day values tend to\nreturn to previously observed during controlled ovarian\nstimulation.\nTable 1 Demographics and assisted reproductive technology cycle parameters between two groups. (Implantation, N = 46 and\nNon-Implantation, N = 123). Descriptive statistics between two groups. Mean values with standard deviation (SD)\nNon-Implantation N = 123 (72.8%) Implantation N = 46 (27.2%) t-Test p value\nFemale Age (in years) 34.94 ± 4.03 (19 –39) 34.28 ± 3.35 (25 –39) 0.290\nMale Age (in years) 36.14 ± 4.76 (22 –46) 37.19 ± 5.91 (29 –62) 0.832\nTime of Infertility (in months) 54.46 ± 33.82 (12 –204) 60.22 ± 38.49 (14 –192) 0.375\nType of Infertility: 0.297\nPrimary 95/123 (77.2%) 38/46 (82.6%)\nSecondary 28/123 (22.8%) 8/46 (17.4%)\nAntiMullerian hormone (pg/mL) 2.45 ± 2.45 (0.09 –16.65) 2.62 ± 2.46 (0.04 –13.56) 0.679\nAntral follicle count 8.43 ± 5.07 (2 –40) 8.63 ± 3.74 (2 –20) 0.801\nTotal dose of gonadotropins (in International Units) 2500.81 ± 812.19 (300 –4500) 2508.15 ± 757.91 (450 –4500) 0.956\nProgesterone levels at Trigger day (ng/mL) 0.88 ± 0.44 (0.01 –2.20) 0.78 ± 0.47 (0.01 –2.10) 0.188\nNumber of collected Oocytes 8.25 ± 5.14 (2 –22) 10.50 ± 5.20 (2 –23) 0.140\nMetaphase II Oocytes 6.57 ± 4.22 (2 –17) 7.06 ± 4.77 (2 –21) 0.150\nNumber of day 3 embryos 3.18 ± 2.40 (2 –12) 3.84 ± 2.65 (2 –12) 0.120\nNumber of blastocyst for vitrification 0.65 ± 1.51 (0 –6) 0.86 ± 1.71 (0 –9) 0.200\nTable 2 Ultrasound parameters between two groups. (Uterine artery resistance index and uterine artery pulsatility index) at baseline,\nat day 6, 8 and 10 after controlled ovarian stimulation, at trigger day and at embryo transfer day. Mean values with standard\ndeviation (SD). rhCG – recombinant Human chorionic gonadotropin\nNon-Implantation N = 123 (72.8%) Implantation N = 46 (27.2%) t Test p value\nUterine Resistance Index (Ut RI)\nBasal 0.97 ± 0.16 0.92 ± 0.12 0.1\nDay 6 1.01 ± 0.15 0.94 ± 0.11 0.04\nDay 8 1.09 ± 0.14 0.97 ± 0.12 0.001\nDay 10 1.19 ± 0.16 1.07 ± 0.16 0.001\nTrigger Day with rhCG 0.93 ± 0.10 0.88 ± 0.09 0.011\nEmbryo Transfer Day 1.12 ± 0.12 1.02 ± 0.09 0.001\nUterine Pulsatility Index (Ut PI)\nBasal 1.46 ± 0.51 1.33 ± 0.34 0,06\nDay 6 1.64 ± 0.45 1.47 ± 0.40 0,023\nDay 8 1.74 ± 0.47 1.44 ± 0.44 0.001\nDay 10 1.87 ± 0.43 1.51 ± 0.37 0.001\nTrigger Day with rhCG 1.48 ± 0.38 1.18 ± 0.27 0.001\nEmbryo Transfer Day 1.91 ± 0.54 1.49 ± 0.42 0.001\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 3 of 7\n\nSubendometrial blood flow analysis (resistance and pul-\nsatility index) showed no statistical difference between\ntwo groups at baseline, with increasing values for both\ngroups during controlled ovarian stimulation. During that\nperiod and also at trigger, and at embryo transfer day,\nthere was statistical difference between two groups with\nlower scores for the implantation Group (resistance index\nof 0.78 ± 0.16 versus 0.65 ± 0.12 with p value of 0.001 and\npulsatility index of 0.95 ± 0.14 versus 0.83 ± 0.14 with p\nvalue of 0.001 for non-implantation versus implantation\ngroup at trigger day respectively) (T able3 and Fig. 2).\nThe ratio between subendometrial blood flow and uter-\nine artery fluxometry showed no statistical difference be-\ntween both groups at baseline for the resistance index.\nStatistical difference between two groups was set after\ncontrolled ovarian stimulation, trigger day with hCG and\nat embryo transfer for the resistance Index. However, in\nterms of pulsatility index, no statistical difference was met\nbetween the two groups except for the trigger day with\nhCG and at embryo transfer day (0.72 ± 0.12 versus 0.68 ±\n0.11 with p value of 0.01and 0.66 ± 0.12 versus 0.70 ± 0.08\nwith p value of 0.018, for the non-implantation versus im-\nplantation group respectively) (Fig. 3).\nIn this study the intraobserver reliability was 0.96. In\naddition, because the same operator performed all mea-\nsurements, in this study there was no interobserver\nvariability.\nDiscussion\nEndometrial receptivity in ART cycle has always been a\nchallenge for physicians that need real time data in order\nto make better treatment options [ 12–15]. Vaginal 2D\npower doppler ultrasound is a non-invasive and a\nrelative inexpensive tool at clinician ’s disposal [ 16]. Sin-\ngle analysis of endometrial pattern at trigger day has\nbeen the most used, with contradictory findings. Also\nsample size of many studies led to conflicting results\nand further investigation in this area has been postponed\nwith the advent of other technologies. In this study we\naimed to address these issues in a wholesome way with\nseveral observations over time with a good sample size\nin order to obtain further data and better knowledge of\nthe working process underlying endometrial receptivity.\nUterine artery fluxometry (resistance and pulsatility\nindex) showed with statistical difference lower values\nin the implantation group in comparison to the non-\nimplantation group. We could also monitor increas-\ningly higher levels during controlled ovarian stimula-\ntion for both parameters. These findings may relate\nto the hormonal status during ovarian controlled\nstimulation and the effects of higher serum estradiol.\nA significant decrease of all parameters for both\ngroups was observed on the trigger day with rhCG,\nwith the recovery of fluxometry parameters at embryo\ntransfer day. The decrease o f both resistance and pul-\nsatility flow may be associated with the rhCG effect\non vascularization, due to its up-regulation effect on\nvascular endothelial growth factors.\nSubendometrial blood flow displayed a similar pat-\ntern with comparable values at baseline and increas-\ningly higher values during stimulation and also a\nsignificant decrease after rhCG administration and re-\ncovery to previous values at embryo transfer. In a\nsimilar pattern values were statistical similar at base-\nline, and significantly different between the two\ngroups afterwards.\nFig. 1 Serial Uterine Artery Resistance and Pulsatility index flow (Mean values). RI – Resistance Index; PI – Pulsatility Index\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 4 of 7\n\nThe ratio obtained between subendometrial flow\nand uterine artery fluxometry parameters showed, for\nall parameters (resistance and pulsatility) and for both\ngroups that values in the subendometrial compart-\nment were sustained and lower in comparison to the\nuterine artery flow (values always under 1). This\nmeans that subendometrial territory has lower resist-\nance to blood flow allowing further and privileged\nvascularization. We could also note that in terms of\nresistance index the values on the implantation group\nwere lower with statistical difference between the two\nsets. In terms of pulsatility, values were also lower in\ncomparison to uterine artery fluxometry, but no sig-\nnificant pattern was met. Since we are dealing with a\nreason between two values (pulsatility index from\nsubendometrial flow and uterine artery), with increas-\ningly higher values until trigger day with rhCG,\nfollowed by a significant drop and recovery afterwards\nat embryo transfer day, this might be an explanation\nto the observed pattern [ 17].\nWe could not refrain to uphold expectation of these\nresults as they show a serial of values, demonstrating\na certain pattern of evolution that one should expect\nfrom a transforming living tissue and its natural\nTable 3 Ultrasound parameters between two groups. (Subendometrial resistance index, subendometrial pulsatility index,\nsubendometrial / uterine artery resistance index ratio and subendometrial / uterine artery pulsatility index ratio) at baseline, at day 6,\n8 and 10 after controlled ovarian stimulation, at trigger day and at embryo transfer day. Mean values with standard deviation (SD).\nSE/Ut – Subendometrial / Uterine Arteries ratio; rhCG – recombinant Human chorionic gonadotropin\nNon Implantation N = 123 (72.8%) Implantation N = 46 (27.2%) t Test p value\nBasal\nSubendometrial Resistance Index 0.77 ± 0.17 0.71 ± 0.17 0.82\nSubendometrial Pulsatility Index 1.16 ± 0.25 1.01 ± 0.26 0.1\nSE/Ut RI ratio 0.80 ± 0.09 0.76 ± 0.12 0.6\nSE/Ut PI ratio 0.73 ± 0.12 0.77 ± 0.11 0.117\nDay 6 after Controlled Ovarian Stimulation\nSubendometrial Resistance Index 0.84 ± 0.17 0.73 ± 0.14 0.001\nSubendometrial Pulsatility Index 1.14 ± 0.20 0.98 ± 0.22 0.001\nSE/Ut RI ratio 0.82 ± 0.10 0.77 ± 0.09 0.04\nSE/Ut PI ratio 0.72 ± 0.14 0.68 ± 0.12 0.132\nDay 8 after Controlled Ovarian Stimulation\nSubendometrial Resistance Index 0.95 ± 0.16 0.79 ± 0.18 0.001\nSubendometrial Pulsatility Index 1.24 ± 0.20 1.03 ± 0.25 0.001\nSE/Ut RI ratio 0.87 ± 0.13 0.81 ± 0.12 0.04\nSE/Ut PI ratio 0.75 ± 0.15 0.73 ± 0.10 0.615\nDay 10 after Controlled Ovarian Stimulation\nSubendometrial Resistance Index 1.04 ± 0.718 0.88 ± 0.19 0.001\nSubendometrial Pulsatility Index 1.32 ± 0.23 1.12 ± 0.31 0.001\nSE/Ut RI ratio 0.88 ± 0.11 0.82 ± 0.08 0.04\nSE/Ut PI ratio 0.72 ± 0.10 0.75 ± 0.11 0.251\nTrigger Day with rhCG\nSubendometrial Resistance Index 0.78 ± 0.16 0.65 ± 0.12 0.001\nSubendometrial Pulsatility Index 0.95 ± 0.14 0.83 ± 0.14 0.001\nSE/Ut RI ratio 0.84 ± 0.14 0.73 ± 0.09 0.01\nSE/Ut PI ratio 0.72 ± 0.12 0.68 ± 0.11 0.01\nEmbryo Transfer Day\nSubendometrial Resistance Index 0.99 ± 0.15 0.87 ± 0.12 0.001\nSubendometrial Pulsatility Index 1.19 ± 0.17 1.07 ± 0.20 0.001\nSE/Ut RI ratio 0.84 ± 0.15 0.74 ± 0.10 0.001\nSE/Ut PI ratio 0.66 ± 0.12 0.70 ± 0.08 0.018\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 5 of 7\n\nadaptations in need to further assist on the complex\nbinding process of implantation.\nConclusions\nEndometrial receptivity plays an important role in the\nsuccessful outcome in ART cycles. Much has improved\nover recent years in the area of embryo transfer, and\nembryo cultures. Yet the underlying mechanism that re-\nsults in failure of implantation of a good quality embryo\non a supposed receptive endometrium is still unclear.\nImplantation window is the most critical period of time\nin human reproduction. In a clinical point of view, prac-\ntitioners need to have some objective measurements to\ndetermine the probability for a healthy pregnancy.\nFig. 2 Serial Subendometrial artery Resistance and Pulsatility index flow (Mean values). RI – Resistance Index; PI – Pulsatility Index\nFig. 3 Serial Subendometrial / Uterine Arteries Ratio – Resistance and Pulsatility index flow (Mean values) RI – Resistance Index; PI –\nPulsatility Index\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 6 of 7\n\nMany techniques have been developed but results are\nstill controversial, or in some cases proven to be too in-\nvasive and lacking reliability especially in women with ir-\nregular menstrual cycles.\nThe continuous evolution of endometrium makes it\ndifficult to establish a pattern that might be useful in\nidentifying a receptive endometrium.\nUltrasound developments have been able to clarify and\nmake aware more information about the morphoky-\nnetics of this tissue and its changes throughout the cycle.\nBetter understanding of the role that makes an endomet-\nrium receptive may be the key in solving these issues,\nproviding a diagnostic tool that will enhance ART cycles\nand elective embryo transfers more effective in produ-\ncing better outcomes.\nThis study showed that endometrial 2D power doppler\nanalysis may identify a receptive endometrium as soon\nas day 6 of ovarian stimulation. Uterine artery fluxome-\ntry and subendometrial blood flow as single evaluation\nparameters, or in combination as a ratio show a clear\ncontinuous mechanism that enables endometrium to be-\ncome receptive to a healthy embryo. In this way clini-\ncians may be made aware of this possibility and further\nenhance its procedures with better knowledge weather\nor not to perform embryo transfer on that given cycle.\nAcknowledgements\nWe would like to appraisal M.S. for the help with the statistical analysis of\ndata.\nAuthors’ contributions\nRSM, AHO and JMO are responsible for the study design. RSM has been the\nprincipal investigator and the principal collector of data. RSM has been\nresponsible for data analysis. DVO, AHO and JMO have been responsible for\nreviewing the article for publication. All authors read and approved the final\nmanuscript.\nFunding\nNo Grant support on this study.\nAvailability of data and materials\nEncrypted non-disclosure data available at Open Science Framework data-\nbase for peer review purpose only. Project name Physical Biomarkers in\nEndometrial Receptivity with access link: https://osf.io/hr25m/?view_only=\n8d5f6dcb8b25420bbd9188382163e7d7\nEthics approval and consent to participate\nThe study has been approved by the Ethics Committee of our Institution\n(CHCB 22/2017). Oral and written consent was obtained for all willing\nparticipants prior to registering for this study. Patient Informed consent to\nparticipate in this study CHCB 22/2017.\nThe authors have consented for publication.\nCompeting interests\nThe authors declare that they have no competing interests.\nReceived: 4 May 2019 Accepted: 25 July 2019\nReferences\n1. Altmae S, Mantinez-Conejero JA, Salumets A, Simon C, Horcajadas JA,\nStavreus-Evers A. 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Ultrasound Obstet Gynecol. 1996;7:135 –40.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nSilva Martins et al. Reproductive Biology and Endocrinology           (2019) 17:62 Page 7 of 7","source_license":"CC0","license_restricted":false}