Methods of improving gamete selection when employing cryopreservation techniques.
\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\\n"}]',published:!0,mainMedia:{caption:"Highly Cited",originalUrl:"/media/original/117"}},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\n'}],latestNews:[{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"},{slug:"introducing-intechopen-book-series-a-new-publishing-format-for-oa-books-20210915",title:"Introducing IntechOpen Book Series - A New Publishing Format for OA Books"}]},book:{item:{type:"book",id:"192",leadTitle:null,fullTitle:"Electric Vehicles - The Benefits and Barriers",title:"Electric Vehicles",subtitle:"The Benefits and Barriers",reviewType:"peer-reviewed",abstract:"In this book, theoretical basis and design guidelines for electric vehicles have been emphasized chapter by chapter with valuable contribution of many researchers who work on both technical and regulatory sides of the field. 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He has authored more than 50 technical papers affiliated to post-doctoral visits to Strasbourg University, France in 2018, Charles University in Prague, Czech in 2014, and Western Michigan University, the USA in 2006. He is a member of different geology and geophysics associations around the world and an award receiver among which is the one for Scientific Excellence.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"102766",title:"Prof.",name:"Khalid S.",middleName:null,surname:"Essa",slug:"khalid-s.-essa",fullName:"Khalid S. Essa",profilePictureURL:"https://mts.intechopen.com/storage/users/102766/images/system/102766.jpg",biography:"Dr. Khalid S. Essa obtained his B.Sc. with honors (1997), M.Sc. (2001) and Ph.D. (2004) in Geophysics from the Faculty of Science, Cairo University. He joined the staff of Cairo University (1997) and was appointed a research Professor of potential field methods in the Department of Geophysics (2014). He has undertaken affiliated post-doctoral visits to Strasbourg University, France (2018-2019), Charles University in Prague, Czech (2014-2015) and Western Michigan University, USA (2006-2007). He has authored more than 70 technical papers and served as an Editor and external reviewer for many top journals. He attended several International Geophysical Conferences in USA, Australia and France. He was a member in SEG, AGU, AAPG, EAGE and EGS. Also, he is a member of the National committee for Geodesy and Geophysics, Academy of Scientific Research and Technology, Egypt (2020-2023) and member of the Petroleum and Mineral Resources Research Council, Sector of Quality Councils, Academy of Scientific Research and Technology, Egypt (2018-2021). He has been awarded the Award of Prof. Nasry Matari Shokry in Applied Geology, Academy of Scientific Research & Technology (2017) and the Award of Cairo University for Scientific Excellence in Interdisciplinary, Multidisciplinary and Future Sciences (2017).",institutionString:"Cairo University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"Cairo University",institutionURL:null,country:{name:"Egypt"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"10",title:"Earth and Planetary Sciences",slug:"earth-and-planetary-sciences"}],chapters:[{id:"79172",title:"Gravity Field Theory",slug:"gravity-field-theory",totalDownloads:98,totalCrossrefCites:0,authors:[null]},{id:"82023",title:"Gravity Anomaly Interpretation Using the R-Parameter Imaging Technique over a Salt Dome",slug:"gravity-anomaly-interpretation-using-the-r-parameter-imaging-technique-over-a-salt-dome",totalDownloads:8,totalCrossrefCites:0,authors:[null]},{id:"81073",title:"New Semi-Inversion Method of Bouguer Gravity Anomalies Separation",slug:"new-semi-inversion-method-of-bouguer-gravity-anomalies-separation",totalDownloads:7,totalCrossrefCites:0,authors:[null]},{id:"78623",title:"Gravity and Inertia in General Relativity",slug:"gravity-and-inertia-in-general-relativity",totalDownloads:132,totalCrossrefCites:0,authors:[null]},{id:"78182",title:"Temporal (t > 0) Space and Gravitational Waves",slug:"temporal-t-0-space-and-gravitational-waves",totalDownloads:120,totalCrossrefCites:0,authors:[{id:"300154",title:"Emeritus Prof.",name:"Francis",surname:"Yu",slug:"francis-yu",fullName:"Francis Yu"}]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"347259",firstName:"Karmen",lastName:"Daleta",middleName:null,title:"Ms.",imageUrl:"//cdnintech.com/web/frontend/www/assets/author.svg",email:"karmen@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"7315",title:"Minerals",subtitle:null,isOpenForSubmission:!1,hash:"f0d5c2a9a5f37e6effcb8486c661d217",slug:"minerals",bookSignature:"Khalid S. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"63467",title:"Clinical Outcomes of Assisted Reproductive Techniques Using Cryopreserved Gametes and Embryos in Human Medicine",doi:"10.5772/intechopen.80627",slug:"clinical-outcomes-of-assisted-reproductive-techniques-using-cryopreserved-gametes-and-embryos-in-hum",body:'\nThe development of cryopreservation techniques has made possible the use of frozen and thawed gametes and embryos aiming at reproduction, by means of assisted reproductive techniques (ART). Cryopreservation allows to banking gametes for later use, including also the possibility to be used by other individuals, as in donor treatments. Effective techniques such as in vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI), worldwide applied, offer a high efficacy by the creation of supernumerary embryos. As recognised downsides of IVF/ICSI treatments include the high prevalence of perinatal complications due to multiple births, the recommended practice of transferring fewer embryos in the fresh IVF treatment cycle, with the goal of performing single embryo transfer and the cryopreservation of remaining embryos for their later use in frozen-thawed cycles, one at a time, is currently the trend [1]. The cumulative chance to achieve pregnancy and live-birth through IVF/ICSI treatments is thus enhanced by the later use of thawed embryos in separate treatments.
\nThe methods for cryopreservation of embryos and gametes have demonstrated effective and safe, and have developed towards the achievement of a clinically established level. These methods are also currently being offered to patients suffering of cancer, due to the risk of infertility associated with certain cancer treatments, or to individuals with conditions that have an inherent risk of premature gonadal insufficiency and infertility, aiming at fertility preservation [2].
\nAlthough embryo cryopreservation has historically been regarded as the first-choice technique for fertility preservation, social, ethical and legal reasons usually restrict its use to couples who have entered into a committed long-term relationship. However, women without a partner may attempt this possibility using a sperm donor. Furthermore, this issue has also been shown to translate to fertility preservation undertaken electively, with one study finding that >80% of patients undergoing oocyte preservation by choice were single at the time, and that lack of a partner was by far the most common reason for not pursuing child-bearing earlier [3].
\nAs such, the cryopreservation of gametes affords individuals an increased level of reproductive autonomy, and ensures that fewer patients are faced with the extraordinarily difficult decision of later reproducing with a partner who may no longer be ideal, or not reproducing at all. Here, the most common indications for gamete cryopreservation in both males and females and embryo cryopreservation will be discussed, along with their clinical outcomes, necessary considerations and future perspectives.
\nAlthough semen cryopreservation has remained an established technique for many years, the cryopreservation of mature oocytes was considered experimental by the American Society for Reproductive Medicine (ASRM) until 2013 [4]. As such, this relatively recent development has paved the way for an explosion of social fertility preservation (‘social freezing’), found by a HFEA report to have increased more than two-fold between 2013 and 2016. In fact, a 10% increase in the number of egg freezing cycles was reported from 2015 to 2016 [5]. In a field which was once dominated by fertility preservation following medical diagnosis, this represents a dramatic paradigm shift, which must be regulated to ensure transparency for, and protection of, the prospective patient. This differs from traditional approaches to non-elective fertility preservation, where patients who may be younger or have no desire to delay childbearing are faced with a high likelihood of complete infertility following resolution of their disease. Therefore, such conditions encompass those that directly cause premature ovarian insufficiency (POI) such as bilateral benign ovarian tumours, severe recurrent endometriosis or genetic disorders (e.g., Turner’s syndrome), and conditions that indirectly result in POI such as malignant or non-malignant diseases that require the administration of gonadotoxic chemo- or radiotherapy [6].
\nWhilst there is a tendency to focus primarily upon female infertility due to the intrinsically finite nature of female reproductive biology, it must be remembered that males are also distinctly susceptible to gonadotoxic agents, with one study reporting that up to 60% of male cancer survivors experience fertility impairment [7]. In fact, malignant diseases are amongst the most significant indirect contributors to infertility worldwide, with some of the most commonly-used classes of chemotherapeutics, alkylating agents, having been shown to induce POI in 42% of women treated [8]. The situation is further complicated by the widespread use of novel targeted therapies whose impact upon fertility is largely unknown [9]. These advances in cancer treatment efficacy (coupled with societal pressures to delay childbearing) have led to an increasing proportion of cancer survivors who wish to further add to their families, resulting in increased public awareness of treatment-induced subfertility, increased demand for fertility-preserving procedures, and the emergence of a brand-new field: oncofertility [10, 11]. This new discipline is badly-needed, providing patients with essential information that will impact upon their treatment decisions and future family planning, and aiming to disrupt the traditional lack of emphasis placed on iatrogenic infertility in the oncological sphere [11, 12, 13].
\nAnother newly-emerging paradigm in gamete cryopreservation is its implementation as a timesaving method in fertility treatment. Age is the most significant determinant of IVF cycle outcome, meaning that older females who present for treatment may be considered for multiple consecutive rounds of ovarian stimulation and egg collection, thereby facilitating the freezing of large numbers of eggs which can later be fertilised and transferred [14]. This is a significant advantage for couples who may want multiple children, or who find the storage of a large number of embryos ethically questionable. It is open to debate whether this application should be considered medical or social, but as technology advances, it is important we consider such applications that lie within the ‘grey areas’ of medicine.
\nSperm cryopreservation is the only established fertility preservation method in post-pubertal males, and has been in clinical use for over 50 years [15]. Its early adoption to the clinical realm is attributed to the accidental discovery of the cryoprotective properties of glycerol on sperm cells, their abundance for experimental uses and their small size. This latter property is an extremely important one, reducing the likelihood of damaging intracellular ice crystal formation during the freezing process. Whilst cryopreservation by slow freezing protocol was the first method used successfully, it causes extensive chemical and physical damage to sperm cell membranes, with only 60% of sperm regaining motility post-thaw [16]. Comparative studies have demonstrated that non-standard methods of rapid freezing (vitrification) using liquid nitrogen give better post-thaw motility rates and alter protein expression profiles less, as well as being more time- and cost-efficient [17, 18]. Whilst both methods result in a significant reduction in viability, the (generally) high number of spermatozoa per sample means that lower survival rates are acceptable. As such, either cryopreservation method may be used effectively. This relatively low bar for post-thaw viability contrast hugely with oocyte cryopreservation, where the numbers of gametes collected tends to be small, and therefore more stringent protocols and attrition rates are required.
\nSperm samples for cryopreservation are usually obtained by masturbation, but if in the cases of azoospermia, males who are unable to provide a sample (e.g., for psychosocial or physical reasons) or those who have previously undergone a vasectomy, surgical techniques may be employed. These include epididymis aspiration, testicular needle biopsy (TESE) or needle aspiration (TESA), with TESE having impressive success rates of 85%, even following chemotherapy for testicular cancer [19]. It is important to note that although these methods of sperm retrieval are effective, all require that the patient is able to produce spermatozoa, even at dramatically decreased levels. Options are extremely limited for patients whose Sertoli cells are non-functional, or pre-pubertal males, with the cryopreservation and autotransplantation of spermatogonial stem cells (SSCs) still classified as experimental, but showing promise in animal models [20]. In vitro maturation of SSCs, or SSC derivation from induced pluripotent stem cells (iPSCs) are also avenues under investigation [21].
\nWhilst both the American Society of Clinical Oncology (and almost all other) guidelines recommend that fertility preservation be offered to pubertal males before commencement of gonadotoxic treatment, only 25% of eligible males in the relevant cohorts bank sperm. These statistics are surprisingly low, especially when one considers the generally non-invasive nature of semen sample collection, and the wealth of prospective studies supporting that viewpoint that the overwhelming majority of men diagnosed with cancer wish to have children later in life [22]. One such study reported that 43% of patients surveyed ranked reproducing as a ‘top 3’ life goal [23]. It is therefore apparent that a disconnect exists in male fertility preservation that is not present to the same degree in the female equivalent. This may be due to routinely poor counselling by clinicians, but it is also possible that the priorities of young male patients may not adequately reflect their later life goals, or that male stoicism might affect the decisions made. Equally, the perceived high cost of cryopreservation and storage might have a role to play, even though robust cost-benefit analyses have shown sperm cryopreservation to be more cost-effective than post-therapeutic fertility management [19]. It has been evidenced that long-time storage does not seem to affect the fertilisation potential of sperm, as recently reported after 40 years of storage [24].
\nIn contrast to spermatozoa, mature (MII) oocytes are large, fragile cells that are much more susceptible to water retention and ice crystal-mediated damage. Furthermore, addition of cryoprotectants may result in osmotic stress, with the cumulative effect of these stressors manifesting as thickening of the zona pellucida, premature cortical granule exocytosis and meiotic spindle disruption [25, 26]. Although this disruption of the meiotic spindle appears to be transient in almost all cases, there is robust evidence to show that cryopreservation negatively impacts oocyte gene expression and proteomics, with some cryoprotectants even shown to alter maternally-derived proteins which support early oocyte development [27, 28, 29]. The net result of this is a ‘stressed’ oocyte which is difficult for spermatozoa to penetrate and fertilise. As such, the clinical applications of oocyte cryopreservation were limited until the inception of the ICSI technique in 1992 [30], with the first pregnancy derived from frozen oocytes following in 1997 [31].
\nAnother quantum leap forward in the efficacy of oocyte cryopreservation came with refinement of freezing protocols. Similar to the paradigm change seen in spermatozoa cryopreservation, vitrification (fast freezing) techniques were pioneered, first producing a live birth in 1999, and then being further improved by Japanese groups in 2003 [32, 33]. In contrast to the small increase in efficacy seen with the introduction of vitrification in spermatozoa cryopreservation, however, vitrification of oocytes seems to greatly increase post-thaw oocyte survival and fertilisation rates, with a 2014 Cochrane review finding a relative increase in oocyte survival of 29%, and a 19% increase in fertilisation [34]. An additional meta-analysis of three RCTs in 2016 reported a 16.1% increase in survival (RR = 1.23, 95% CI: 1.02–1.49; P = 0.031) [35]. The efficacy of the vitrification technique was further confirmed when a large prospective study of Spanish egg-donation programmes could not detect any statistically significant difference between using fresh donor eggs, when compared to vitrified frozen eggs [36]. It is important to note, however, that both of these techniques are inherently operator-dependent; with vitrification especially variable due to the need to complete the process within seconds [37]. This is an important caveat, and highlights the importance of training and upskilling, especially when considering the variable experience that operators may have within the same fertility clinic. It must also be clarified that the survival rates of oocytes (and the number collected) are likely dependent on the age and disease status of the donor, meaning that the extremely high survival rates of thawed oocytes reported by some studies on donor eggs (in excess of 96%), may not be truly representative for a significant proportion of patients who undergo fertility-preserving treatment [38].
\nIt is clear, therefore, that the path to the clinic for oocyte cryopreservation has not been a straightforward one, with the early, highly-ineffective methods of oocyte cryopreservation making it an unrealistic and imprudent option for females in urgent need of fertility preservation, such as oncology patients. Cancer in reproductive age is twice as common in females as in males, and more than half of those diagnosed are expected to undergo treatment that compromises their fertility [39]. One large retrospective study highlighted this, indicating that whilst the incidence of treatment-related acute ovarian failure (AOF) was approximately 10%, these figures greatly misrepresent the total age-specific impact on fertility, with 40% of those not reporting AOF encountering infertility by the age of 35 [40]. Furthermore, the probability of early menopause was ‘at least’ 25% by age 30 [40]. It is likely, therefore, that effects on fertility may often relate to a reduction in the overall number of primordial follicles, and may therefore remain undetected until later in life. In a society where increasing numbers of women are choosing to delay childbearing, this may mean that women who are presumed to have normal reproductive activity following resumption of menstruation may not try to conceive as early as they are able to, and then later encounter difficulty.
\nIt is important to consider that patients undergoing fertility-compromising cancer treatments may only have sufficient time for one round of ovarian stimulation and egg collection before their treatment must begin. This process of controlled ovarian stimulation (COS) followed by egg collection generally takes approximately 2 weeks to complete, with patients able to start chemotherapy within 48 h of completion. Whilst concerns had initially been raised about the administration of such high doses of exogenous gonadotrophins to patients with hormone-sensitive cancers (e.g. breast, ovarian), effective and safe stimulation protocols using aromatase inhibitors have been developed and shown to result in no increased risk of recurrence in breast cancer, after a mean 5-year follow-up period [41]. In addition, the use of GnRH antagonist regimens (in place of the usual GnRH agonist regimens) allow ovarian stimulation to be started at any point in the menstrual cycle (‘random-start protocols’), thereby minimising treatment delays. These GnRH antagonist regimens have been shown to result in the collection of similar numbers of mature oocytes and produce similar fertilisation rates [42]. Moreover, they have been shown to result in a lower risk of ovarian hyperstimulation syndrome (OHSS) than conventional protocols [43].
\nAs such, refinements in cryopreservation techniques and stimulation protocols represent incredibly important steps for cancer (and elective) patients, increasing both the safety of oocyte collection and the likelihood of a live birth following completion of treatment.
\nAs outlined above, the cryopreservation of gametes is a technically difficult and expensive process. As such, it is essential that the true success rates of these procedures be analysed using clinical endpoints, in order to prevent delays to treatment, unnecessary harm to patients and to disrupt the growing belief amongst the general proportion that egg freezing constitutes an infallible ‘insurance policy’ against age-related fertility decline.
\nIn order to assess the success of cryopreservation we must first examine the parameters by which success is gauged. The most realistic way to evaluate the efficacy of cryopreservation techniques (and indeed individual clinics) is through the comparison of live births achieved per oocyte thawed. Although this may seem obvious, there is a growing propensity for some clinics (especially those who derive a significant proportion of their income from social egg freezing) to display these statistics in a manner that makes them appear more impressive. For example, some success rates might be represented using clinical pregnancy rates per thaw cycle; with some studies reporting this to be as high as 78% [44]. This figure is not an accurate representation of the reality faced by most patients, with the largest reported study of 3610 vitrified oocytes producing an oocyte survival rate of 90%, translating to a clinical pregnancy rate of 48% and an ‘oocyte-to-baby’ rate of just 6.5% [45]. If this same study were to be presented alternatively, it could be quoted as a delivery rate of 78.8% per oocyte donation cycle. As such, it is clear that there must be further efforts to homogenise how ‘success’ is calculated, and increased scrutiny of how these results are presented to potential patients. It is essential, also, to note that this data (and indeed almost all data on oocyte cryopreservation) has been generated from oocyte donation programmes. This is significant because oocyte donors tend to be carefully-selected, young individuals, whose eggs are likely to be of greater quality than the average patient wishing to engage in autologous fertility preservation. In fact, this viewpoint is supported by findings that only 32% of patients freezing their eggs were below the age of 35, and recent data showing reduced yield of oocytes collected in oncology patients versus matched controls [5, 38, 44]. As such, it is likely that the true likelihood of a successful live birth for patients in these groups is significantly lower than the figures generated by current data. It is essential, therefore, that the increasing availability of data from non-donation sources be interpreted and used to validate the statistics that are currently quoted.
\nThe largest study using data collected from outside of egg donation programmes was carried out by Cobo et al., who examined the reproductive success of 1468 women undergoing elective oocyte cryopreservation for non-oncologic reasons [46]. Their data clearly demonstrates the impact of age at freezing upon potential success, with those who froze at or before the age of 35 having a 53.9% likelihood of a live birth per ET, whilst those freezing at or above the age of 36 had a 22.9% chance. This viewpoint was echoed by a recent HFEA report, who described patient age at freezing as ‘the most important factor’, whilst age at thaw was not determined to have any statistically significant impact [5]. The same study also demonstrated the importance of the number of oocytes obtained to vitrify in increasing chanced of a live birth, with an increase from 5 to 8 oocytes producing the most significant increase in LBR (8.4% per oocyte if <35). Whilst an average ‘oocyte-to-baby’ ratio is omitted, it is estimated to be significantly lower than the 6.5% achieved in donor programmes. Whilst this is an interesting figure, it is likely that it does not provide as clear a picture of the factors that impact oocyte viability as that provided by age-bracket stratification.
\nConsequently, we can conclude that the number of viable oocytes available for fertilisation is a clear determinant of the likelihood of successful pregnancy. The technique used to freeze and thaw the oocytes retrieved is thus of the utmost importance, with a multitude of studies confirming the advantages provided by vitrification protocols, both in terms of post-thaw oocyte survival and reported pregnancy rate. In fact, multiple studies reported the clinical pregnancy rate (CPR) to more than double when compared to slow-freezing protocols [47, 48]. In addition, there is increasing scrutiny on the impact that the rate of warming can have on post-thaw oocyte survival and characteristics. In fact, Mazur and Seki reported oocyte survival >80% when ultra-rapid warming was carried out, even when using traditional slow-freeze protocols. Further expanding on this, they demonstrated that such methods could be used to reduce the concentrations of cytotoxic chemoprotectant required for the vitrification process [49]. Interestingly, a recent meta-analysis of five studies concluded that there was no significant difference between the fertilisation rates, embryo cleavage or pregnancy rates achieved when using fresh versus vitrified oocytes [50]. This viewpoint is supported by recent data supplied by the HFEA, who concluded that the birth rate per embryo transfer (PET) was rising to over 19%, and within 2% of the overall IVF birth rate PET [5]. In addition, multiple studies have demonstrated that the length of storage has no effect on pregnancy rates or outcomes [45, 51]. As such, it is reasonable to conclude that cryopreservation techniques have advanced to such a stage that significant future improvements in success rates will likely relate to methods of increasing the yield of oocytes collected per stimulation cycle, or in the methods used to select the embryos to be transferred.
\nThe above discussion is necessarily focused on female gametes, as spermatozoa quality has traditionally been seen to be of less importance owing to the large number usually obtained per collection and their high survival rate. It is also worth noting that studies have found no correlation between sperm quality and disease stage in oncology patients [52]. In addition, the advent of ICSI has meant that even ‘poor quality’ sperm samples with low motility scores can be used to produce a viable embryo. That said, it is almost certain that there exist intrinsic variations in spermatozoa quality that currently evade detection, driving increased research into how we select the sperm we use for fertilisation, both in the context of conventional IVF, and in fertility preservation. The artificial techniques discussed have abrogated the physiological selection methods inherent to the natural reproductive process, paving the way for a growing need for the ‘unnatural selection’ of favourable gametes via novel biomarkers or growth characteristics. Ongoing avenues of such research include the assessment of spermatozoal DNA fragmentation rates (although evidence is not yet conclusive), and promising future avenues such as the stratification of sperm quality via spectrophotometric analytical techniques such as Raman spectroscopy [53]. In fact, the latter method would allow andrologists to select spermatozoa on the basis of both their homeostatic and epigenetic context [54].
\nIt follows that an essential aspect of any discussion on the clinical outcomes of gamete cryopreservation must be that of perinatal outcomes. It is often easy to rely on pregnancy rate as the sole benchmark of a successful preservation cycle, but serious consideration must also be given to whether the progeny created are morphologically, genetically and developmentally ‘normal’. Reassuringly, a number of analyses, one of 165 pregnancies and another of 936 infants, have found a comparable incidence of congenital abnormalities in infants born following oocyte vitrification, conventional IVF and natural pregnancy [55, 56]. There is also a growing body of evidence, however, that IVF may trigger epigenetic disruption in the developing embryo, potentially causing the slightly lower birth weights observed amongst children born as a result of these techniques [57]. That said, it is also possible that these differences are related solely to the increased ages of the patients within the IVF cohort. A long-established relationship exists between increased parental age and genetic dysfunction, with increased maternal age being linked to abnormal meiotic spindle function, and therefore the induction of gross chromosomal abnormalities such as Trisomy 21 (Down’s syndrome) [58]. Similarly, it has been shown that the higher prevalence of single point mutations seen in children born to fathers of more advanced age is attributable to the higher number of mitotic replications that these germ cells have experienced [59]. It is thought that this is a direct cause of the increased rates of neurodevelopmental disorders, leukaemias and stillbirths seen in this paternal cohort [60]. As such, although age has a strong positive correlation with adverse perinatal outcomes, no cryopreservation-specific (or indeed fertility treatment-specific) causal relationship has yet been reliably established.
\nIn fact, the most common perinatal outcomes that are directly attributable to IVF are due to multiple pregnancies. These usually occur as a consequence of the transfer of more than one embryo, and may result complications such as premature birth, intrauterine death and conversion to caesarean section [61]. Whilst this, and complications associated with advanced maternal age, certainly remain considerations in the fertility preservation sphere, the patients concerned tend to have fewer options and less time to achieve a successful pregnancy, making the delivery of multiple children more serendipitous than it otherwise might be. Indeed, as the average age of childbearing increases (due, in part, to ART), it is arguable that discussion of such ‘difficult pregnancies’ will be of less future importance, as prospective patients will almost always opt to try to conceive in the face of an increased risk of poor perinatal outcome, instead of not attempting to conceive at all.
\nIn conclusion, although the oocyte conversion rates discussed above might seem extremely poor at the outset, it must again be stressed that modern assisted reproduction technologies circumvent the physiological selection mechanisms that serve to ensure only the most viable gametes survive. Success rates using cryopreserved gametes are almost comparable to those achieved using fresh gametes, and therefore it is reasonable to expect the efficacy of both techniques to advance in parallel as our knowledge and gamete selection methods improve. The Table 1 presents several methods currently used to improving gamete selection for cryopreservation.
\nTechnique | \nDescription | \nEvidence | \n
---|---|---|
Pre-freeze swim-up preparation (spermatozoa) | \nTraditionally, sperm selection via preparative techniques was undertaken post-thaw. There is increasing evidence, however, to show that such swim-up techniques should be performed before cryopreservation to produce the highest percentage of viable spermatozoa. It is theorised that cytokine release from immune cells that are inadvertently included in cryopreserved samples may damage spermatozoa quality, and that this could be avoided using these pre-freeze techniques. This viewpoint has been supported by data from recent trials. | \nPetyim et al. [111] | \n
Rate of cooling | \nAs evidenced by the aforementioned increases in gamete quality using vitrification techniques, the rate of cooling during cryopreservation is extremely important. As such, efforts have been made to dramatically decrease the volume of the solution in which oocytes are vitrified (now 0.1–2 μL). To facilitate this, specialised carriers have been developed, including both open and closed systems. Comparative analysis of these two categories of systems has demonstrated similar oocyte survival rates, but significantly increased cytoplasmic vesicle presence (and theorised reduction in quality) in oocytes frozen using the closed system. | \nBonetti et al. [112] | \n
Low-CPA protocols | \nProtocols that employ low concentrations of cryoprotectants have the potential to combine the positive aspects of vitrification and slow-freezing, without their respective associated disadvantages. Although such protocols have been impractically complex and time-consuming, recent advances in quartz micro-capillary techniques are showing promise. | \nChoi et al. [113] | \n
Single-gamete analysis | \nAlthough not yet adequately optimised for clinical use, analysis of individual gametes has the potential to revolutionise how ART is carried out. The increasing need for artificial selection has meant that there is now increasing scrutiny on spectrophotometric and other non-invasive analytical techniques, some of which have been shown to provide adequate comparative analysis for oocyte quality and sperm DNA fragmentation rate. Whether this comparative analysis will be of clinical use, however, remains to be seen. | \nDavidson et al. [114] Sanchez et al. [115] | \n
In-vitro maturation (IVM) of immature oocytes | \nIVM aims to increase the yield of oocytes available for cryopreservation through the obtaintion of additional M2 oocytes from oocytes that would otherwise be discarded. Although data shows that approximately 35% of IVM oocytes can produce cleavage-stage embryos when fertilised, and this method has been suggested to increase the efficacy of treatment cycles aimed at fertility preservation, there is currently insufficient data to support the systematic use of IVM techniques or the freezing of immature oocytes. | \nOktay et al. [116] Phoon et al. [117] | \n
Selection via DNA fragmentation rate | \nIt is clear that a both vitrification and slow-freeze protocols produce DNA lesions, either via full or partial fragmentation. Although modern analytical techniques can quantify this fragmentation (and resulting apoptotic induction), they most commonly result in destruction of the gamete in question. As such, although they may provide valuable information on the quality of a particular sample, they do not provide a solution for the accurate selection of gametes which may prove more viable that their morphologically-normal counterparts. | \nValcarce et al. [118] | \n
CPA equilibration temperatures | \nChanging the equilibration temperature with CPA and increasing the sucrose concentration added have both proven to be effective strategies to improve oocyte survival and fertilisation rates, respectively | \nBorini et al. [119] | \n
Methods of improving gamete selection when employing cryopreservation techniques.
Although the technical aspects of gamete cryopreservation have been discussed at length above, one must also consider the societal and ethical impact of such procedures. Gametes are incredibly prized cells; holding the genetic information is required to produce related offspring for those at high risk of fertility disruption. Therefore, the conditions under which they are stored, the individuals permitted to handle them, access related information whilst they are stored, and the length of time which they can be stored for are of the utmost importance. Although legislative circumstances may vary from country to country, the HFEA permits storage of gametes or embryos for an initial maximum period of 10 years, with this being extended by 10 years at a time on a case-by-case basis up to a maximum of 55 years [61]. These limits are important to protect the wellbeing of prospective children, and to prevent the misuse of genetic material. Furthermore, as technological advances in genetics allow increasingly accurate prediction of phenotype and disease likelihood, it is likely that the genetic material contained within gametes will need progressively more stringent protection. An example of such measures includes the recently-enacted General Data Protection Regulation (GDPR), which legislates for the prevention of the misuse of such genetic data [62].
\nThe societal effects of the growing popularity of cryopreservation must also be considered. More women than ever before are experiencing the ironic dichotomy of spending the vast majority of their reproductive years trying no ensure that they do not fall pregnant, but then finding themselves unable to conceive when they try to. As such, the landscape of this exploding field is increasingly commercial, providing increased funds to facilitate advances in treatment efficacy at the cost of advertising cryopreservative services as an insurance policy against age-related fertility decline. There is also a worrying increase in the number of companies offering ‘social freezing’ as part of their employee benefit packages. This is a trend that propagates the misinformed idea that social cryopreservation guarantees a later pregnancy, and serves to perpetuate the societal pressure placed on women to delay childbearing [63]. The cost of such procedures (if not covered by insurance or a third party) is also a valid consideration, with various cost benefit analyses finding contrasting conclusions on whether it is more, or less cost-effective to cryopreserve in one’s mid-twenties and return to them at age 40, or just to attempt conventional IVF at age 40 [14, 64]. Whilst this is an important avenue of discussion, the superior success rates provided by the cryopreservation route are likely to provide a superior chance of obtaining a live birth.
\nWhile the risks associated with childbearing at an increased age may have the immediate downstream effects of reducing the incidence of certain genetic aberrations, it is also important to consider knock-on effects which may not be immediately obvious. It is possible that widespread societal gamete cryopreservation could unearth harmful novel ART-mediated epigenetic alterations, or further promote the delay of childbearing age. Such effects would doubtless affect the composition of our society, and the manner in which it functions. Therefore, the future direction and regulations governing this area must be scrutinised to determine what should, and should not be permitted. This is a more complex ethical discussion that falls outside of the scope of this chapter, but should nonetheless be kept in mind.
\n\n | Slow freezing | \nVitrification | \n
---|---|---|
Cleavage stage embryos | \n1.5 M PROH plus 0.1 M sucrose are included in the most commonly used protocols [74] | \nEG-based method was early proposed for vitrification of cleavage stage embryos [122] | \n
DMSO-based method initially reported the lowest survival rate [123] | \n||
EG/DMSO/sucrose in open or close systems are the most commonly used cryoprotectants | \n||
Blastocysts | \nGlycerol and sucrose as cryoprotectants are included in the most commonly used protocols [74] | \nEG and DMSO were the cryoprotectants used in the first pregnancy reported after blastocyst vitrification [124] | \n
EG/DMSO/sucrose in open or close system are also used [97, 125] | \n
Commonly used protocols for cryopreservation of cleavage stage human embryos and blastocysts.
Abbreviations: EG: ethylene glycol; DMSO: dimethylsulphoxide.
\n | Cleavage stage | \nBlastocyst stage | \n||
---|---|---|---|---|
\n | Day 2 | \nDay 3 | \nDay 5 | \nDay 6 | \n
Morphology before cryopreservation [74, 81, 97] | \n≥4 blastomeres | \n≥6 blastomeres | \nBlastocysts are scored according to expansion, inner mass and trophectoderm using Gardner scoring system – 3BB or better | \n|
<25% fragmentation | \n||||
No multinucleate blastomeres | \n||||
Morphology after thawing [74, 120] | \n≥50% intact blastomeres | \nScoring according to Gardner, as before cryopreservation | \n||
Higher number of blastomeres after 24 h of culture | \n||||
Expected survival [74, 121] | \n61.4–87.5% with slow freezing | \n76.3–88% with slow freezing | \n||
64–94% with vitrification | \n84–100% with vitrification | \n
Morphological aspects of embryos before/after cryopreservation and expected cryosurvival.
Since the early days of in vitro fertilisation (IVF) 40 years ago, there have been remarkable advances in clinical and laboratory areas that have opened the door to different variants of standard IVF procedure [65, 66]. Improvements of ovarian stimulation protocols enable the collection of several mature oocytes, which associated with the improvement of the IVF techniques and optimization of embryo culture result in the obtention of a large number of embryos. Therefore, embryo cryopreservation was a necessary evolutionary step for IVF-treatments with the first pregnancy after transfer of a frozen-thawed embryo being reported in 1984 [67]. Since then, embryo cryopreservation has become a widely used technic in assisted reproductive technology (ART), allowing the preservation of the remaining embryos following a fresh transfer for future pregnancies and as a modern tool to reduce multiple births by encouraging patients to transfer a single embryo [1, 68]. Additional indications for embryo cryopreservation are the embryo banking for preimplantation genetic screening, elective deferred embryo transfer, when the patient is at risk of a hyperstimulation and for fertility preservation [66, 69]. Thus, embryo cryopreservation greatly increased the safety and efficacy of IVF treatments and enable the later use of all the embryos obtained from a single oocyte pick-up.
\nOver the years, cryopreservation methods, protocols and stage at time of cryopreservation have changed, improving embryo cryopreservation techniques. Consequently, the number of frozen-thawed cycles increased worldwide [66, 70] with similar or even higher pregnancy rates compared with the transfer of fresh embryo [65, 70]. In Europe, the last report generated from registers by the European Society of Human Reproduction and Embryology (ESHRE) stated that 154,712 frozen-thawed cycles were performed in 2013, increasing the overall life birth rate by 6% [71].
\nHerein we will resume the evolution of the embryo cryopreservation methods, stage at which cryopreservation is performed and give an overview of the perinatal outcomes of frozen-thawed embryo transfers.
\nSince the first reports of pregnancy and delivery after transfer of frozen-thawed embryos in the earlies 1980s [67, 72] various protocols of embryo cryopreservation were introduced. They mostly differ from each other in the type and concentration of cryoprotectants, equilibration timing, cooling rates and freezing devices [35]. Regardless of the cryopreservation method used, the goal is to suspend embryos in time by cooling embryos from ambient temperature to −196°C [73]. Nowadays slow freezing and vitrification are the two principal approaches for embryo cryopreservation, although vitrification has become favored over the last decade [35, 74].
\nIn slow-freezing protocol the temperature is decreased sufficiently slowly to allow the adequate cellular dehydration but also minimising the formation of intracellular ice. This is only possible through the use of a programmable freezing machine. With this method, the samples are first exposed to a quick cooling rate of 2°C/minute until they reach −7°C. Then extracellular ice crystal formation is induced manually (seeding) by touching the vial or straw with precooled forceps as far away from the embryos as possible. As consequence, more water leaves the embryo allowing cryoprotectants to enter. After the seeding, the temperature decreases slowly (0.3–1°C/minute) untill it reaches temperatures approximately −40°C and then rapidly to −150°C with a cooling rate of approximately 50°C/minute. The embryos are then stored in liquid nitrogen until use [35, 74].
\nAlthough the first pregnancies and birth were obtain with an embryo cryopreserved with dimethylsulphoxide (DMSO) as cryoprotectant [67, 72] births using other cryoprotectants, such as propanediol (PrOH) were soon reported [75, 76]. Since then, this has become the cryoprotectant more widely used in combination with sucrose for embryo cryopreservation by slow-freezing [35] (Table 2). The disadvantage of the slow-freezing method is the formation of ice crystals, increasing the risk of cell damage during thawing. Therefore, despite this method has being used for over 30 years in IVF laboratories and considered safe, since the concentrations of the cryoprotectants used to avoid ice crystal formation are low, another technique was developed to improve embryo cryopreservation—the vitrification.
\nWith vitrification, the ice formation is almost eliminated since the cells and the extracellular milieu are solidified into a glass-like state [77]. This method has an extremely high cooling rate in the range of 2500–30,000°C/minute till −196°C by immediate exposure to liquid nitrogen [74, 77]. Despite the high concentrations of cryoprotectants that this method requires, its potential toxicity is reduced by the short time of exposure and the small volume of cryoprotectants used [35]. As in the slow-freezing method different cryoprotectants were tested, leading to the current preferred combination of DMSO (15%), ethylene glycol (EG-15%) and sucrose (0.5 M) in a minimum volume (≤1 μl) [78] (Table 2). The biggest difference between vitrification protocols relates to the cooling and storage methods employed, with open system, involving direct embryo contact with the liquid nitrogen, or closed system involving specific devices to avoid direct contact with the liquid nitrogen [35].
\nWith vitrification a laboratory can expect to obtain an increased embryo cryosurvival rate comparing to the slow-freezing method (Table 3), has well as a beneficial effect in the clinical pregnancy rate and live-birth rate per embryo transfer [35, 74, 79, 80]. Additionally, vitrification method does not require any specific equipment and is less time consuming compared to slow-freezing. Consequently, many laboratories worldwide have completely replaced slow-freezing with vitrification [35].
\nThe first pregnancy was obtained with an embryo cryopreserved at eight cells stage [67]. Since then the procedure has changed several times, with the current practice being to preserve either at the cleavage stage Day 2 or 3 of culture or at the stage of blastocyst at day 5 or 6 of culture, despite no clear evidence of which strategy is more beneficial for frozen-thawed embryo transfer [81, 82]. Since only few randomised controlled trials (RCTs) have been conducted to determine what stage of development optimises cumulative birth rate for the retrieval cycle, most of the available data about the timing of embryo cryopreservation is derived from outcomes of fresh cycles [73].
\nDespite the method of cryopreservation and embryo’s stage, the embryo selection for cryopreservation is based on their morphology and pre-freezing morphology is directly related with cryopreservation success and efficiency [83].
\nFor cleavage stage embryos, it is recommended that the embryos selected for cryopreservation should have 4 cells at day 2 and 8 cells at day 3, less than 10% of fragmentation, stage specific cell size and no multinucleate blastomeres [84] (Table 3). After thawing, embryos with 100% intact blastomeres will have a higher implantation. However, in embryos with 50% or more cells intact post-thaw and with mitotic resumption, the number the cells at the transfer may be more predictive of the embryo’s ability to implant than the percentage of cells surviving at the time of thawing [85, 86, 87]. These parameters remain the most clinically important criteria to evaluate the implantation rate potential till today [83].
\nAdvances in culture systems have made possible to prolong embryo culture until the embryo reaches the blastocyst stage. Thus, over the last decade blastocyst transfer at day 5/6 of culture has greatly increased and is seen for some as a “natural selection” of the most viable embryo, similar to the process during spontaneous conception [88, 89]. However, the clinical efficacy of blastocyst transfer over cleavage stage transfer is debatable. In fact, in 2016 a Cochrane meta-analysis reported an increase of clinical pregnancy and live birth after blastocyst transfer [82] but 1 year later another meta-analysis did not find any statistical difference in outcomes when comparing the transfer of embryos at the cleavage stage or blastocyst [90], the same results were previously described for cryopreserved embryos [81].
\nAs with cleavage stage embryos, assessment of blastocyst stage cryopreservation outcomes requires attention to variety of factors before cryopreservation and after thawing in addition to methodology. Outcomes have been shown to be dependent on pre-freeze quality of the blastocyst and time required to reach the blastocyst stage [91, 92].
\nThe most commonly used blastocyst grading systems assigns scores to three morphologic aspects of the embryo: quality of inner cell mass—grades A, B, C; quality of the trophectoderm—grades A, B, C and degree of expansion (Table 3). Blastocyst score at the time of cryopreservation was associated with survival and implantation rates [93, 94, 95]. Other factors that may contribute to outcomes are the day the cryopreserved embryo reached the blastocyst stage [5, 6], whether the blastocoel was collapsed or not prior to cryopreservation, and evidence of blastocoel re-expansion prior to transfer [96, 97].
\nFrozen-thawed cycles increased during the last decade and a concern about the perinatal outcome also have risen [66]. Although embryo cryopreservation is a well established procedure, long-term studies are still sparse [35]. Data are reassuring suggesting that pregnancies obtained from frozen embryos are not associated with an increased perinatal risk compared to fresh transfers [98, 99, 100, 101, 102, 103, 104]. Several reviews have indicated a slightly better result when frozen-thawed embryos were used compared to the fresh transfer, with reduced risks of preterm birth, small for gestational age babies, low birth weight babies and pre-eclampsia, which could be justified by the endocrine milieu of the stimulation when the transfer is made fresh [70, 99, 102, 104].
\nFurthermore, a systematic review, published recently, confirmed that singleton babies born after the transfer of frozen-thawed embryos have higher weight at birth when compared to babies born after the transfer of fresh embryos, as well as a higher risk of hypertensive disorders during pregnancy [70].
\nTo further improve IVF outcomes, it has been suggested to freeze all the embryos obtained in a stimulation cycle and then plan a deferred transfer during a natural cycle or with hormone replacement with exogenous estradiol (E2) and progesterone (P) for endometrial priming [105, 106]. With this strategy, the frozen-thawed embryos are transferred into a more “physiological milieu” which seems to improve implantation and outcomes compared to fresh transfer [106, 107, 108]. However, almost all the data was obtained in patients with high ovarian response patients and thus it was suggested that the freeze all strategy should be perform on patients with a risk of ovarian hyperstimulation syndrome (OHSS), since it was not clear if normal and poor responders will be the same benefits from freezing all the embryos [109].
\nA recent retrospective study using the general population has reported that 50.74% of patients using the freeze-all strategy achieved a live birth after the first complete cycle [105]. Additionally, another study indicated positive results in poor ovarian responders, and suggested the freeze-all strategy as an alternative to cycle cancellation for these patients [110]. Despite these positive results, large multi-centre randomised controlled trials are needed to evaluate the freeze-all strategy [105, 110].
\nIn conclusion, the cryopreservation of gametes and embryos is a rapidly developing field that demonstrates increasingly comparable success rates to those encountered in conventional IVF using fresh gametes or embryos. Aiming to provide reproductive autonomy for patients, it is intrinsically intertwined with both societal and ethical issues, and will doubtless play an increasingly central role in how we as a species reproduce over the coming decades. Research indicates also safety of reproductive treatments using cryopreserved gametes and embryos.
\nK. Rodriguez-Wallberg is supported by research grants from Stockholm County Council, Karolinska Institutet, The Swedish Cancer Society and Radiumhemmets’s Research Funds.
\nTrophic relationships between organisms are the mechanisms responsible for most of energy and nutrient transfers; they allow the functioning of the ecosystem. These relationships, known as food webs, caught the attention of naturalists before the concepts of evolution and ecology were about to be determined.
Initially, the diet of a species and its skills to obtain it were recognized as the leading factors for the prevalence of the fittest. Additionally, it is one of the main forces leading to evolution of that species in the long term [1]. Furthermore, competition for food became one of the favorite hypotheses to explain species exclusion; it states that when two species seemed to feed on the same resources, the best suited ultimately leads its competitor to extinction in the long term [2]. This idea has been around for many years and has not been completely discarded or proved [1].
Examining phototrophs, also known as primary producers, is the dominant starting point to analyze food webs. They use the incoming sun’s energy and inorganic nutrients to generate their biomass. This is the most important mechanism, as it initiates the cycling of nutrients and energy flux in aquatic food webs. There is primary productivity involving chemolithotrophs dominating in places devoid of sun’s light [3]. These places were mostly known to be, until recent times, around underwater volcanoes more than 1000 meters deep [3, 4].
Primary production is at the base of all consumers concurring in the environment. However, macroscopic food webs tend to be very short, with few levels of consumers because these organisms dissipate matter and energy efficiently [5]. All metazoans invest their energy looking for food, ingesting it, digesting, repairing themselves, mating, and reproducing. These activities make multicellular organisms to get around 10% of biomass fixation efficiency. Thus, 1,000 kgs of the primary producer will be needed to produce 100 kgs of herbivorous animals, only 10 kgs of small carnivores, 1 kgs of medium-sized carnivores, and only 0.1 kgs of top carnivores, following a pattern known as pyramid of productivity [5]. Adding a predatory species at any level would destabilize the food web, as this will consume higher amounts of biomass [6]. Energy dissipation is even larger, meaning that the entropy produced during the functioning of the food web is very high. However, only 1% of the incoming sunlight is used for primary production, stressing the importance of the environmental factors limiting biomass productivity to sustain food webs.
Primary productivity varies along seasons. When it reaches its peak, productivity is controlled by the top predator’s consumption (top-down), and when it reaches its lowest level, productivity is controlled by phototrophs (bottom-up). There are places that are permanently bottom-up controlled such as the deep ocean communities depending on the “organic matter rain” from dead organisms living in the photic zone in places near the equator are almost always top-down controlled, where productivity may be at its peak for most of the year. All other places experience top-down/bottom-up controls alternatively, depending on the productivity seasons.
Unicellular algae lead primary productivity in marine environments, sustaining the great diversity of organisms, especially in places receiving nutrient inputs from lands. Heterotrophic unicellular organisms forage on algae and both phototrophs (phytoplankton) and heterotrophs (zooplankton) conform to the plankton. However, unicellular organisms span in sizes less than 1 μm to hundreds of micrometers, and the species’ diversity of plankton, including microbial eukaryotes and bacteria, ranges in the order of thousands. Species of microorganisms are much more numerous than the metazoans. With such a great diversity of microorganisms, it become apparent that the microbial food webs may function differently from the macroscopic food webs.
It was believed that food webs would get destabilized if the number of species increases at any level above the primary producers. However, microbial food web seemed to get more stability with the increasing number of species, contradicting what was observed in macroscopic food webs [7]. Thus, the higher number of species of bacterial and microbial eukaryotes in aquatic food webs seemed to contradict that assumption; this phenomenon was named as “The paradox of microbial loop.” It was paradoxical that productivity and efficiency of nutrients and energy transformation is increased by adding more species, promoting the stabilization of the food web [8].
It’s been a long road since the recognition of the “paradox of the microbial loop” in the aquatic food webs. Nowadays, it is referred only as the “microbial loop,” after being integrated into the food web conceptualization in both terrestrial and aquatic environments [7].
The complexity of microbial food webs needs to be approached from the analysis of different functioning capacities and nutritional needs of the participating species. It has been normal to assign very general feeding habits to protists and metazoans, like bacterivores for example. This nomenclature implicates that a single species of protist can feed on any one or indistinctly on all the thousands of bacteria species. However, observation of feeding habits has revealed that protists and metazoans prefer feeding on specific kind of bacteria while avoiding other species. Pigmented bacteria [9], for example, has fewer predators than non-pigmented ones. On the other hand, there are several species of protists, mostly amoebae, small flagellates and
One explanation for pigmented bacteria to have fewer predators relied on the toxicity or poisonous effect of those pigments for many protists, pointing out the importance of the biochemical warfare that bacteria must synthetize to defend themselves. However, chemicals used for evading enemies attract other ones looking for those same compounds, putting bacteria in a situation where there is no way out for bacterial preys. Indeed, there is no way out of being preyed upon, as every living being has predators, or at least other species which may feed on them or use them as a resource.
Is there a single factor determining the feeding preferences? The short answer is “No.” Remember that “bacterivorous” or “algivorous” are labels used to recognize the kind of food that protists and metazoans may prefer to feed on, and it involves many species. From the beginning, this was a non-exclusive way to label the category of food that may be used to group the highest quantity of species to simplify and conceptualize the food webs. Furthermore, during the first half of the XX century [11], there were many very interesting studies trying to determine the “diets” of several species of protists [11, 12], with the aim of designing a chemically defined culture media, as is the case of several recipes for culturing
Designing a culture media for protists or bacteria was a major task, as numerous factors about their nutritional needs were unknown (and remain unknown). These attempts to cultivate bacteria and protists lead to one important conclusion: different species cannot synthesize one or several molecules needed for their metabolism and have to take those molecules, as such, from their ingested food [12] or from other microorganisms that live within the biofilm (such as the case of NAD+ **, which the bacteria has to consume from other species of bacteria for both of them to grow). Microbial biologists named this phenomenon as “auxotroph” [13]. In this way, the molecule(s) a bacterial species is auxotroph for must be added to the culture media, to keep a culture of such species [14]. The kind of molecules, their diversity, and their macro- and micronutrient composition form a universe comparable to the one containing the species’ diversity on the planet.
Ecological relevant functions have been recognized in prokaryotes and microbial eukaryotes. Bacteria have been cataloged as nitrogen fixing, denitrifiers, metanogens, methanotrophs, phosphorous mobilizers, metal mobilizers, phototrophs, and chemolithotrophs as the main recognized functions in the ecosystem. On the protists’ side, several trophic groups have been recognized as phototrophs and phagotrophs. The first group is strictly divided between the phototrophs and mixotrophic ones, while the second one may be divided in bacterivorous (including cyanobacteria), frugivorous (feeding on hypha and or yeasts cells), algivorous, protist consumers (raptorial protists), and metazoan predators. Parasitic bacteria, pathogenic bacteria, and microbial eukaryotes have been largely studied from the medical point of view. However, recently, they have been studied from the ecological perspective (their impact on the predator–prey relationships, the “health” of species populations protected for conservation, and their effect on the nutrients distribution along food webs [13].
Phagotrophic protists may ingest very different kinds of particles and present the capacity to eject the ones they cannot digest, or even reject particles previously ingested [15]. Even if the water current would transport a good mixture of different bacterial species, phagotrophs may choose which particles ingest and eject the debris from their digestion together with the non-digestible microorganisms. This means that protists may show preference for the kind of food they most likely can digest (recognizing their preys by their quorum sensing signals), and, like bigger organisms, they may need a variety of food sources to get the nutrients they need [15].
A close examination of the different trophic groups allows to re-mark the unicellular phototrophs as the most productive in terms of biomass production since there is no synthesis of support or conductive structures, and, because of that, they are the base of the aquatic food webs.
The phagotrophic protists have been recognized for being the main consumers along microbial trophic networks in aquatic systems conforming a major proportion of the microbial biomass in these systems [16, 17]. These predators are also responsible for much of the recycling flow of nitrogen and phosphorus in the aquatic systems [18].
Particularly the ciliates are key elements of aquatic food webs they have several functions, they can be primary producers, predators, they serve as food for metazoans including free-living stages of metazoan parasites; there are many aquatic habitats without macro-organisms, but none without bacteria and at least few protist species [19].
One of the most interesting groups of protists are the mixotrophic ones. Some of them may correspond to the old morphological groups of ciliates, flagellates, and ameboebas. Mixotrophy is defined as the ability to combine phagotrophy and phototrophy in a single cell [20]. This group can be divided into constitutive mixotrophs, meaning they have the innate ability to photosynthesize, and the facultative or non-constitutive mixotrophs. These organisms may sequester the plastids after consuming their phototrophic preys or by harboring photosynthetic endosymbionts [20, 21]. Around 23% of planktonic ciliates species (marine and freshwater combined) perform acquired phototrophy, and this ability is present in at least 8 main ciliated taxa: Heterotrichea, Hypotrichia, Oligotrichida, Stichotrichida, Litostomatea, Prostomatea, Peniculia, and Peritrichia. Phototrophy is usually acquired from algae endosymbionts in 7 of these 8 ciliated taxa. Contrastingly, Oligotrichida usually obtains this ability by plastid sequestering [22].
The structures of the mixotrophic ciliates community varies through seasons, depending on the changing water trophic condition. Mixotrophic ciliates dominate in spring and summer, reaching from 58–100% of the ciliates in oligotrophic waters [23, 24, 25], but represent only 5% of the total community of ciliates in winter, probably due to the lower water temperatures and nutrients. These conditions restrict the growth of algae, negatively affecting the population of mixotrophic ciliates if their preferred species of algae is missing [24].
The mixotrophic ciliates are mainly from the genera
Ward and Follows [33] performed a global simulation of the ocean-surface food web, revealing that mixotrophy enhances the transfer of biomass to larger organisms at higher trophic levels, which in turn increases the efficiency of oceanic carbon storage through the production of larger and faster sinking conglomerates of organic molecules. It follows that mixotrophic protists play a key role in modulating the primary production that underlies the food web in aquatic systems [21, 22, 32]. However, their importance has not been fully appreciated because traditional field and laboratory studies focus on strict classifications as phototrophs or phagotrophs [32] because incorporating this flexibility to acquire food is difficult to modelize. Mixotrophy is known to be common in all aquatic systems but its contribution to net community production is difficult to quantify, and the integration of their impact on the global biogeochemical cycles remains to be incorporated.
Ciliates and flagellates are the most dominant bacterivores among the phagotrophic protists in most aquatic systems [16, 34], consuming between 25–100% of the daily production of marine phytoplankton together with large quantities of bacterial biomass [18]. Bacterivores and algivorous protists are the core consumers of microbial biomass in aquatic food webs [16, 17] regulating these groups in two apparently contradictory ways: by feeding on the abundant food source, they keep in check their further expansion, that in turn gives other less preyed species the opportunity to become more numerous, and at the same time, the release of cellular wastes (from protists) enhance the reproduction of the species being predated. The combined effect of these two processes enhances the nutrient cycling and fuels biomass productivity. By performing this activity, ciliates and flagellates increases their own biomass, attracting metazoan predators and functioning as linkage of lower and upper trophic levels in aquatic food webs [16, 35, 36].
The size of the ciliate determines the sizes of preys they can feed on. Thus, bacterivorous ciliates ingest a different particle size range; the preferred size spectrum for each species is a function to cytostome size and morphology. For example, small ciliates usually bacteria eat <3 μm [18, 37, 38]. Ciliates that feed on the smallest particles (<1 μm) require relatively high densities of these bacteria as a minimum to keep their population growth [30]. Several groups of ciliates actively feed on specific bacteria species for a period ranging between 44% and 100% of the time, because bacterial densities will have variations as responses to predation intensity along time [36].
Bacterivorous ciliates are present in all aquatic environments, from oligotrophic to eutrophic, in both freshwater and oceans. The diversity of bacterivorous ciliates and their contribution to the flow of energy in trophic networks depend on the dynamics of the systems in which they are living. Therefore, food resources are probably the main regulators of ciliated communities (diversity, abundance, and biomass) [30]. For example, bacterivorous ciliates contribute very little for the direct transfer of bacterial production to the trophic networks of metazoans in oligotrophic environments. Ciliates consume less than 11% of bacterial productivity in these waters [39, 40, 41]. Perhaps the heterotrophic bacteria that are very small in these lagoons (0.035 to 0.4 μm) are grazed by bacterivorous ciliates at a very low rate [41], or the number of bacteria is not enough to support larger ciliate communities feeding on smaller bacteria (<1 μm), as they require high densities of bacteria to maintain their populations [30]. Then, productivity of oligotrophic systems function most of the time as bottom-up (availability of substrate and nutrients) controlled [42]. This functioning will remain until seasonal pulses of nutrients (or human subsidies) arrive, busting primary productivity and changing the system into top-down control, and it will keep functioning the same way until the pulse of nutrients (or subsidy) is completely metabolized, returning the system to the bottom-up dynamic.
Contrastingly, densities of heterotrophic bacteria in eutrophic environments are sufficiently higher to also keep a higher diversity of active bacterivores [43], fueling ciliates biomass productivity and allowing the intervention of metazoan predation. Top-down control (predation) seems to be in function all the time for regulating the abundance of heterotrophic bacteria in eutrophic systems [42]. Normally, communities of bacterivorous ciliates of small sizes (~ 30 μm) are found as dominant in eutrophic environments [30, 38]. The most abundant ciliates in these environments are small oligotrichs (
Sessile ciliates such as
Trophic groups | Examples | References |
---|---|---|
Bacterivores | Colpodida ( | [34, 46, 48, 114, 116] |
Feeding on Phototrophs | Choreotrichia ( | [54, 56, 57, 58, 60] |
Predators of predators | Heterotrichea ( | [62, 64, 66, 68, 72, 108] |
Omnivorous | Choreotrichia ( | [49, 66, 69, 73, 79] |
Mixotrophos | Litostomatea ( | [23, 25, 26, 28, 29, 31] |
Trophic groups free-living ciliates in aquatic environments.
There is a difficulty in assessing a proper name for the kind of food protists feed on when they become predators of phototrophs, as this group consists of both eukaryotic and procaryotic members, and neither of these primary producers may be considered as “plants” or “herbs”. Feeding on them cannot be considered as herbivory. On the procaryotic part, cyanobacteria are a phylum comprising many species that, besides being phototrophs, can also produce toxic molecules, compromising the fresh water supplies for human consumption when growing unchecked in oligotrophic waters [50, 51]. From the eukaryotic part, there is an extra complication when trying to separate the permanent phototrophs from the mixotrophs.
Moving the sizes up, ciliates are one of the most important groups feeding on phytoplankton in marine and freshwater environments [18, 41, 52]. They may consume up to 74% of the daily phytoplankton production [53], becoming the key controllers of phytoplankton biomass [54]. On the other hand, ciliates mobilize the highest proportion of organic carbon and nutrients in oligotrophic waters dominated by cyanobacteria, playing the fundamental role of linking the productivity of microbial food web with the metazoans [41, 53]. It has also been noticed that the flux of carbon up to metazoans is not interrupted when the density of bacterivores ciliates falls, but it is compensated by predation on ciliates feeding on phototrophs [41]. Some of the ciliates that feed on phototrophs are in Table 1.
Ciliates feeding on phototrophs represent between 30–65% of the total biomass of all functional groups of ciliates thriving in eutrophic lakes [55]. However, this dominance is not permanent. Ciliates feeding on phototrophs become very numerous on the blooming season [56], and even dominate the entire ciliate community for short periods between seasons [57].
Tintinnids tend to feed on small-cell-sized phytoplankton (2–20 μm) [58]. They are voracious phytoplankton feeders that may consume over half the quantity of these kind of phototrophs in marine waters [54] and over 69% of these primary producers in lakes [59]. Species like
Selective feeding has been observed in several species of ciliates. However, feeding on a wider spectrum of sizes and kind of phototrophs (non-selective feeding) allows them to take advantage of the productivity in hypereutrophic environments rich in small particulates [49]. The genus
There are several species of ciliates and flagellates that feed on bacterivorous protists and on protists feeding on phototrophs. These are predators of predators. These predator species may feed temporarily on bacteria but cannot survive by just this consumption; they are attracted to them as they offer clues to discover their preferred preys: ciliates, flagellates, or amoebae feeding on bacteria.
Most of predator ciliates feed on preys around 10 times smaller than them [62, 63], although raptorial feeders may consume bigger preys, comparable to their own size or even bigger [64]. This capacity is due to their very flexible cytostome as is the case in protostomatids genera
Predatory ciliates are present in small numbers along seasons in oligotrophic waters, showing surges in population numbers, in synchrony with the increase of primary productivity during the spring, reaching up to 55% of the total ciliates’ abundances in temperate waters [64, 66]. However, they only reach between 24.6% to 28.7% in freezing oligotrophic waters of the Arctic and Antarctic [67].
On the other hand, predatory ciliates become important top-down controllers of microbial food web productivity in eutrophic and hypertrophic waters [68]. Eutrophic waters have the conditions to sustain high productivity rates of phototrophs and heterotrophic bacteria, sustaining, in turn, large populations of their grazers, promoting the increase of predatory ciliate population [69]. Biomass of raptor ciliates may reach almost an order of magnitude higher in eutrophic compared to the one obtained in meso and oligotrophic lakes, suggesting that they are effectively controlling the primary productivity [70]. This assumption is supported by the covariance of predatory ciliates and their preferred food. For example, the increasing population of predatory ciliates bigger than 100 μm is related to a simultaneous shrinkage of abundance of smaller ciliates (<20–40 μm), mostly phototrophs and bacterivorous [71]. Big and voracious ciliate raptors like
Several species of oligotrichs feed on bacterivorous flagellates, showing an efficiency of 45% biomass transformation, also fueling the bacterial productivity by releasing essential nutrients for heterotrophic bacteria to keep their population growth [65]. Some predatory ciliates are shown in Table 1.
Omnivorous protists are an important group to look for when assessing the stability of a food web because their very presence means productivity is enough to non-specialists, to feed on a variety of resources. Omnivores strengthen the resilience of planktonic communities by regulating the trophic dynamics [73]. Omnivorous ciliates may have a preferred prey but can easily move to other kinds of prey, which may be more abundant or easier to catch [74]. This variety of resources for true omnivorous ranges from bacteria, algae, other ciliates of different sizes to fungi [73]. This versatility gives them an advantage to withstand a resource limitation by having alternative prey [70]. Additionally, omnivorous ciliates increase the stability of planktonic communities by feeding on species that may pass undetected from their specialized predators, by having densities small enough to get an advantage of the elimination of their competitors and increase their numbers. In this situation, omnivores would prevent them to reach high densities too fast, giving time for their specialized predators to increase to population levels that may effectively control the newly abundant prey.
Omnivorous ciliates are present in any kind of environment allowing the stability of protist communities. They are elements of marine and freshwater ecosystems, both oligotrophic [66, 75] and eutrophic [69, 76], as well as in polar waters [67].
As with the other trophic groups, omnivores show seasonal bursts of abundances in the communities they are part of, especially in oligotrophic waters where they are scarce most of the time, except for occasional bursts [77, 78]. Omnivorous ciliates are commonly found in lakes throughout the year, normally with low species richness, representing between 2–12% of the ciliates species [67, 79]. Their low contribution to the number of individuals makes them reach a peak of 35% during productivity bursts [66, 79]. However, this proportion may steadily increase in the proportion the environment is turning into the eutrophic condition, increasing the species richness, although the densities of omnivorous ciliates may momentarily diminish with the eutrophication [69] as result of the species increase (more species and lower number of individuals by species). Once the eutrophication reaches a steady state, the biomass of the omnivorous ciliates will reach high values and even dominate among ciliates [76].
The numbers of small omnivorous ciliates usually dominate in meso oligotrophic environments, feeding on dominant bacteria (<2 μm) and algae (2–20 μm) [49]. Food concentration is a very important factor, strongly affecting an easily detectable feeding behavior of omnivorous ciliates [73]. Several of the most common omnivorous ciliates are shown in Table 1.
Functionality alone has its own complexity in food webs because, for example, mixotrophs would be functioning as phototrophs or as heterotrophs along different hours during the same day (How long do they function as phototrophs? How long do they the function as heterotrophs?). An extra dimension in this world comes from the different sizes of preys corresponding to the predators’ sizes and the number of cells each individual predator must get to produce another individual [80]. This is one of the reasons why plankton has been divided in microplankton, nanoplankton, and picoplankton. Each category corresponds to the range sizes of microorganisms. The smaller ones like picoplankton and nanoplankton, performing primary productivity (chemolithotrophs or phototrophic [3], can sustain their corresponding predator’s size and be up to ten times bigger, namely nanoflagellates and microflagellates. These are the two groups of protists related to their size and morphology rather than their taxonomic affiliation [81], since very few information is known about them apart from 18S SSU rDNA sequences; they have been recognized performing predatory activity on phototrophs of the smaller sizes.
One alternative to conceptually reduce the complexity of microbial food webs is analyzing them as nested compartments. This means that the transfers of matter and energy takes place inside each compartment corresponding with one size class of producers and its predators because these organisms function in the same time frame. Then, several of these compartments may get integrated in a bigger one by predation of the next size class. Time frame for this bigger class is also bigger than the previous one, as the sizes of the organisms are also bigger and so on. Every compartment of bigger sizes function as concentrator of biomass and disperser of energy. However, the wastes generated in each compartment releases the nutrients once fixed in the biomass fueling the nutrient cycle in compartments of all sizes. Up to here, it looks like the aquatic food web is functioning as a continuum along and through the water column and surface. However, there is a chance of recognizing boundaries to help a better understanding the food webs dynamic.
When hearing the word “boundary”, immediately, the existence of physical barriers delimiting something in space comes to mind. Because of that, it is hard to imagine an aquatic food web being physically limited because our experience has shown us the big animals feeding on all planktonic organisms at once, which could be in thousands or even millions. However, it just represents a small appetizer for a whale.
A careful examination reveals that very small organisms live faster than ones at the immediate upper-sized scale and intuition tells us that time may be experienced in different ways, depending on the size of organisms involved. The size ranges occupied by ciliates in the microbial food web spans from less than 10 μm to more than 4500 μm [82]. Comparatively, their pool of size ranges would be like the pool of sizes from small fishes to whales. Why are these sizes important? Because it can be argued that the velocity of nutrient exchange is faster in the smaller organisms and the nutrients may be “sequestered” for long periods by the bigger and long-lasting animals. In this way, a complication of time arises when trying to diagram the nutrient cycle in the microbial food web. Time becomes another varying feature rather than a constant in food web dynamics. In this way, time may draw the boundaries between compartments and, at the same time, could be avoiding contradicting the nested compartment proposal in the physical limitless aquatic system.
It is easier to recognize physical boundaries in terrestrial ecosystems as the environment changes at slower velocities than the very dynamic aquatic environment. Soil is a heterogenic environment, the opposite to the aquatic ones. It is an environment that cannot be seen through and be dived in. Soil matrix is composed of a very complex mixture of mineral particles, organic matter and living organisms. This mixture is organized in aggregates that may facilitate or resist water and air passing through it but, most importantly, these aggregates proportionate spaces where all living beings can move through soil.
At a microscale, soil aggregates divide the open spaces in two types, the fast water passing by (the space between aggregates) and the slow motion of water in the space inside the aggregate, and consequently of slow-moving air too, as air and water move through the same spaces). These are the soil’s physical boundaries, and this is the environment where roots move and look for hotspots of nutrients, as well as places where microbial symbionts may be found (normally inside soil aggregates). Water reaching soil aggregates dissolved salts and polar molecules that may contain nutrients that will be taken by roots, mycorrhiza, or bacteria. This is a complementary start of plants primary productivity, because plants have to take water from soil together with other nutrients to produce a wide range of molecules, from non-protein forming amino acids to scents and pheromones, as result of what is known as the “secondary metabolism.” Plant primary productivity comprises both photosynthesis-respiration (primary metabolism) and secondary metabolism, irrespective of being vascular or nonvascular.
Soil productivity is dependent on the nutrient exchange velocity rather than the gross amount of bioavailable nutrients. Nutrients used and released very fast means energy is being captured, transformed, and degraded very fast, implying the activities of all participating organisms are taking place so fast that production of biomass at all levels is gaining momentum and its control may come only from consumption (top-down) no matter that nutrients exist in limited quantity. This feature also explains why the smaller organisms can sustain productivity of the biggest ones. In other words, aerial part of plants are very important for primary productivity because it is the place were light, inorganic carbon, and water are used to produce organic molecules that are at the base of primary productivity (Sun’s energy fixation in organic molecules).
Without diminishing photosynthesis’ importance, most of terrestrial plants gather a “productivity teamwork” inside and around their roots, involving mycorrhizal fungi and mutualistic bacteria, a functional place known as the rhizosphere. Almost 80% of the known terrestrial plants need the association with a mycorrhiza, to appropriately complete their life cycle, but all plants need mutualistic bacteria to grow. Microbial partners are indeed an important part of primary productivity, as they actively participate in the acquisition, modification, and metabolization of many organic molecules containing the elements we call “Nutrients.” For example, it has largely been demonstrated that mycorrhiza translocate phosphorus to plants. At present, very few people challenge this. However, what form of phosphorus is translocated from mycorrhiza to plant? Surely, it is not the phosphorous as molecule, but organic molecule where P is forming part of the structure. Plants can take up P from inorganic molecules in general or from phosphoric acid. Why do they need mycorrhiza to supply P? It is still an open question, but the degree of specificity of the plant-mycorrhiza association allows to conjecture that plant and mycorrhiza share metabolites containing nutrients (not just P) for metabolic complementation, and the same could be true for mutualistic bacteria. This would explain why one species of mycorrhizal fungi is mutualistic to several plant species but functions as pathogenic or parasite to other ones.
Contrary to what happens in waters, soil fungi and bacteria are scattered through soil and physically constrained to available surfaces. If they keep growing unchecked, bacteria may become effective nutrient competitors to plants, as nutrients forming bacterial biomass are non-available to plants. Mycorrhiza may move farther away from the root than bacteria and can establish a mutualistic relationship with other roots (whether they are from the same plant or from a different species, it does not matter) to avoid becoming competitors. Absence of bacterivores is a needed condition for bacteria to become a plant competitor in the rhizosphere [83, 84]. Bacterivores ciliates, flagellates, and amoebae release nutrients trapped in bacterial biomass, stimulating both plant and bacterial growth. In the first case, nutrient release allows roots to take them in and bacteria microcolonies may grow again in the root surfaces, already cleaned out, and obtain nutrients from predators’ wastes [84].
Soil’s physical constrains allow growth of bacteria and fungi in differentiated places. Sometimes bacteria also grow on the surface of hypha, helping fungi to mimic bacteria and somehow escape from fungal predators. It has been possible to observe protists feeding predominantly on fungi and avoiding bacteria as much as possible (
This differentiation of soil’s physical spaces makes it easier to visualize the small productivity compartments around roots, absorbing hairs inside small soil aggregates, bigger compartments covering aggregates on the tip of the root and getting in contact through fungal hypha.
Motility of bigger protists are limited to litter and upper soil layer by the available spaces, restricting their abundance in the underneath layers. Testate amoeba, ciliates, and flagellates, around 100 μm, dominate in these 2 layers and actively participate nutrient recycling from litter, while smaller size ciliates like
Primary productivity in soil is restricted to the upper layers where cyanobacteria and eukaryote algae may survive and even form thin layers known as microbial soil crusts. Both phototrophic bacteria and algae may form stable mutualistic symbiosis with other organisms, like fungi, to develop thicker structures composing soil crusts showing lichens and mosses. Beneath and into soil crusts, ciliates, flagellates, and amoebae are among the most important microbial predators, active mainly during the time of water availability [85, 86]. However, the main photosynthetic carbon input is released by roots into soil layers [87]. Roots secrete amino acids and other complex organic molecules to attract symbiotic bacteria and mycorrhiza conforming the trio of soil productivity sustaining microbial food webs deep into soil [88, 89]. Consequently, protists’ species diversity may be higher around roots and the dominance of ciliates may be restricted to the sizes of soil pores [86, 90, 91, 92]. Soil protists were recognized as purely bacterivorous because fungi feeding protists may transitorily feed also on bacteria. However more detailed studies have recognized species of soil protists feeding only on bacteria or fungi [93, 94, 95]. Among the main bacterivorous ciliates are Colpodida (
Fungi and bacteria normally use different kind of organic molecules, bacteria normally metabolize low molecular weight organic molecules while fungi normally metabolize complex organic polymers of high molecular wight [97]. This metabolic difference allows to conceptualize two pathways for nutrient cycling: the bacterial and the fungal paths. However, this concept is being challenged because of the abundance of protists feeding on both kind of microorganisms [98, 99]. All the early recognized fungi feeding ciliates and amoebae in soil ranges from 50 microns to above 150 μm [100]. However, there are also smaller ciliates and flagellates feeding on both spores and hypha [100]. The main groups of specialized fungal feeder ciliates are grouped in the family Grossglockneriidae [93]. This family of ciliates may account for mora than 2% of the protists sequences in the forest litter and grassland while may drop below 0.3% in peatland soil, probably due to the reduction of soil pore sizes [100]. Although, counting techniques based in MPN calculated around 200 cells/gram soil DW in previous studies [101]. Protists have a very limited capacity to disperse throughout the soil system by themselves. However, oligochaeta disperse them as cysts farther than a few centimeters, in the range of several meters both horizontally as well as vertically into the soil system.
Soil functioning is much more variable than the aquatic systems, as it is regularly subjected to dryness and several flooding events per year. For microbial ecologists, soil is a natural stressed environment, having enormous variations of water availability through seasons, especially in arid and semiarid environments. However, there is a comparable situation, although at lesser degree, in the tropical dry forests, temperate, and tundra regions. Even at the equator, the rainy forests may show an excess of soil in water, stressing microbial food webs.
Microbial communities have been evolved by modifications and adaptations in responses to natural stresses that finally allow them to get along with environmental change. The problem we are facing now resides in the velocity of environmental changes imprinted by human activities. The most important, but hardly the only one, resides in the use of fossil fuels because of the acceleration of climate change. The CO2 released as byproduct of combustion is just one of the causes of climate modification in the short term (in historical and geological times). Internal combustion engines also produce other greenhouse gases such as NxO or NO2, having a bigger capacity of keep heat, and this is a big problem generated only for the atmosphere. Hydrocarbons pose a permanent threat of contamination to aquatic and soil systems near the extraction zones, the transporting infrastructure to refineries, infrastructure for later transportation as fuel to expending places, and by illegal activities damaging oil ducts.
Soil microbiota react in different ways along the gradient of contamination when hydrocarbons reach soils. The plume of contamination normally eradicates the phototrophs and exert a strong selective pressure on bacteria and fungi, by killing or inhibiting the growth of sensitive species while enhancing the growth of resistant ones. These effects can be modified by the toxicity of the different compounds rupturing and/or changing the connections of the trophic networks [102, 103].
The effect of hydrocarbon contamination and others contaminants (pesticides, heavy metals) on communities will depend on the intensity, duration, and frequency of the perturbation. Then, lower species richness and abundance, shortening of the trophic webs, and the simplification of the trophic web are among the first observable damages contamination cause on microbial and protist communities [104]. Protists must at least tolerate the presence of the contaminant to achieve this function. Protists do not feed on hydrocarbons, but their grazing activity on the microorganisms that can keep the metabolization of the contaminant as high as another limiting factor allows them to.
Greater richness and abundance of ciliates species are associated with less perturbed areas; the greater the perturbation, the lesser species richness and abundance [105], regardless of the nature of the perturbing factor. For example, a significant reduction of ciliate diversity has been found in systems polluted by high hydrocarbon concentrations [106]. Medium concentrations only reduce the quantity of individuals from dominant species [106], while low concentrations produce an increase in the numbers of heterotrophic protists [107]. Saline accumulation forces the ciliates’ diversity to decrease as salinity values increase [108, 109]. In the same way, acidic pollution produces lower species richness and abundance as the environment becomes more acidic [110, 111], and the same pattern is observed with heavy metals’ contamination [104, 110].
Addition of organic matter in excess suddenly changes the base of production of the microbial food web, from phototrophs’ productivity to heterotrophic bacteria and yeasts’ productivity. The time of reaction is also different along the different microbial groups surviving the contamination event. Bacteria may start their biological activities several hours after the pollution event, whereas yeast and protists will delay from days to weeks, depending on the size of the organism.
Changes of primary producers from phototrophs to heterotrophs scale to functional groups, accommodating species richness and abundance of bacterivores protists, followed by omnivores. This is due to hydrocarbons stimulation of bacterial growth and the consequently increase of bacterivores species [112, 113]. Some species of genera
An increase in diversity and complexity of food webs are direct effects of these perturbations. Oil spill in deep waters increase the richness of the microbial community species and the complexity of their corresponding relationships, and the oil stimulated microbial activity supports greater variety of ciliates functioning along several trophic levels [117].
Other events of enriching oligotrophic systems with organic matter produce similar changes in the community structure of ciliates. Tirjaková and Vďačný [118] analyzed the changes in the communities of ciliates before and after a windstorm hit a stream, and they found a significant increase of ciliates’ species’ richness and abundance after the storm. Several weeks later, the community of ciliates presented the typical values of oligotrophic sites. The increase in resources availability is the factor indirectly responsible of these changes of ciliate community, but later, communities tend to return to states similar to the initial ones after resources exhaustion, which my take place around six months [118]. However, Shabarova et al. [119] report that the microbial community recovers from perturbation to a pre-flood state within two weeks after the event.
Regarding the connections’ shrinkage of the trophic networks, a gradual narrowing of the planktonic size spectrum has been reported in hypersaline lakes, correlated to salinity increases during the summer, resulting in a simplification of the community represented by the ciliated
Communities’ characteristic of hypersaline lakes are dominated by
Regarding the perturbances in the soil ciliated communities, similar effects have been described as in aquatic ecosystems. Exposure of ciliate communities to heavy metals induces a reduction in the biomass of ciliates and this effect lasts for 20 weeks [124]. Insecticides also generate a decrease in ciliates species immediately after contamination, they also generate a change in the dominance of ciliates, the bacterivores (
Protists in general, and ciliates in particular, play a key role in nutrient cycling and food web functioning in both aquatic and terrestrial ecosystems. In the world experiencing climate change and other kind of anthropogenic menaces, protists may be useful partners to tell us how aquatic and terrestrial systems are dealing with these issues while mesmerizing the observer with their great diversity of beautiful forms.
The authors declare no conflict of interest.
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Integrity - We are consistent and dependable, always striving for precision and accuracy in the true spirit of science.
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\n\nIntechOpen is a dynamic, vibrant company, where exceptional people are achieving great things. We offer a creative, dedicated, committed, and passionate environment but never lose sight of the fact that science and discovery is exciting and rewarding. We constantly strive to ensure that members of our community can work, travel, meet world-renowned researchers and grow their own career and develop their own experiences.
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Among them are those associated with pollution, resource extraction and overexploitation, loss of biodiversity, soil degradation, disorderly land occupation and planning, and many others. These anthropic effects could potentially be caused by any inadequate management of the environment. However, ecosystems have a resilience that makes them react to disturbances which mitigate the negative effects. It is critical to understand how ecosystems, natural and anthropized, including urban environments, respond to actions that have a negative influence and how they are managed. It is also important to establish when the limits marked by the resilience and the breaking point are achieved and when no return is possible. The main focus for the chapters is to cover the subjects such as understanding how the environment resilience works, the mechanisms involved, and how to manage them in order to improve our interactions with the environment and promote the use of adequate management practices such as those outlined in the United Nations’ Sustainable Development Goals.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/39.jpg",keywords:"Anthropic effects, Overexploitation, Biodiversity loss, Degradation, Inadequate Management, SDGs adequate practices"},{id:"38",title:"Pollution",scope:"\r\n\tPollution is caused by a wide variety of human activities and occurs in diverse forms, for example biological, chemical, et cetera. In recent years, significant efforts have been made to ensure that the environment is clean, that rigorous rules are implemented, and old laws are updated to reduce the risks towards humans and ecosystems. However, rapid industrialization and the need for more cultivable sources or habitable lands, for an increasing population, as well as fewer alternatives for waste disposal, make the pollution control tasks more challenging. Therefore, this topic will focus on assessing and managing environmental pollution. It will cover various subjects, including risk assessment due to the pollution of ecosystems, transport and fate of pollutants, restoration or remediation of polluted matrices, and efforts towards sustainable solutions to minimize environmental pollution.
",coverUrl:"https://cdn.intechopen.com/series_topics/covers/38.jpg",keywords:"Human activity, Pollutants, Reduced risks, Population growth, Waste disposal, Remediation, Clean environment"},{id:"41",title:"Water Science",scope:"