Characteristics of semen obtained by electroejaculation from various South American species.
\r\n\tCases of Corrosion in PA industrial equipment and plants are presented and discussed, based on the author's experience and knowledge.
\r\n\r\n\t
\r\n\tA singular application is the manufacture of artificial apatite for coating on stainless steel (SS) orthopedic implants in the human body.
South America, the fourth largest continent, presents an extreme geographic variation that contributes to its large number of biomes, which includes the marine rainforests, alpine, deserts, savannahs, grasslands, chaparral, desert scrub, fresh water, marine, and deciduous desert. Due to such biome diversity, the South American fauna consists of various unique animals.
In the past decade, a rapid and continuous decline in mammalian species has been documented, indeed affecting South American countries. Therefore, the worry put on the development of conservation strategies, as well as on the rapid expansion of the commercial interest on wild species around the world, requires an equally rapid development and adoption of assisted reproductive techniques (ARTs). These techniques allow the conservation and multiplication of genetic valuable individuals, as well as facilitate the transport of the germplasm among distant regions. However, the application of ARTs is still a challenge, since data related to basic reproductive information is scarce, and it is indispensable for the genetic management of rare species. Because of the high diversity in reproductive mechanisms among mammals, it is not possible to directly apply protocols developed from one species to another, hindering the extrapolation of developed ARTs. In this sense, this chapter highlights the importance of applying ARTs for the South American wild mammals, showing the most recent studies in this area and the perspectives for its use in conservation programs.
Technology of male gamete consists of various forms for sperm processing, since its collection until its storage by cooling or cryopreservation. In wild animals, the gamete recovery is generally conducted by electroejaculation (EEJ) [1]; nevertheless, for animals unable to ejaculate, or immediately postmortem, one possibility is to obtain epididymal sperm or testicular tissue, which can be cultivated and cryopreserved [2]. After collection, samples could be stored in cryobanks, which maintains the sperm quality for an indefinite time, providing valuable genetic material to be used for ARTs destined to the wild mammals’ conservation and multiplication.
The EEJ is a safe and effective method for semen collection, especially in wild animals, as it allows handling live animals under anesthesia. It consists of a controlled electrical stimulation of the ejaculatory reflex. A transrectal probe coupled with a specific voltage-producing unit [1] is used to apply the stimulus. The types and disposition of the electrodes, the electric stimulation protocol, and the anatomical characteristics of each species exert important influences on the efficiency of the method [3]. Due to the large species diversity, it is necessary to establish adequate species-specific stimulation protocols using appropriate anesthetic procedures, according to animal responses and well-being [4]. Spermatozoon from different species is presented in Figure 1.
Sperm images of some animals in Latin America, (A) Spix’s yellow-toothed cavy—Galea spixii, (B) Coati—Nasua nasua, (C) Collared peccary—Pecari tajacu, (D) Six-banded armadillos—Euphractus sexcinctus.
In general, most researchers use adaptations of the original protocol developed for felid collection [5], consisting of a total of 80 electrical stimulations divided into three series: 30 stimuli (10 stimuli, 2 to 4 V, series 01), 30 stimuli (10 stimuli, 3 to 5 V, series 02), and 20 stimuli (10 stimuli, 5 and 6 V, series 03), with a 5-minute interval among series. This serial EEJ protocol was adapted for various South American felids such as the ocelot (Leopardus pardalis), the margay (L. wiedii), the tigrina (L. tigrinus) [6], the jaguar (Panthera onca) [7], and other carnivores such as coatis (Nasua nasua) [8] (Table 1) and maned wolves (Chrysocyon brachyurus) [9]. In addition, this protocol has been adapted for other animals such as sloths [10], giant anteaters [11], six-banded armadillos (Euphractus sexcinctus) [12], tapirs (Tapirus bairdii) [13], and agoutis (Dasyprocta leporina) [14] (Table 1).
A totally different serial protocol was described for primate species. For spider monkey (Ateles geoffroyi), stimulation series begins at 1 V, followed by 1 V increments up to a maximum of 10 V. Each series lasts 5 minutes, with 3-minute rest periods between series [15]. For capuchin monkey (Sapajus apella), stimulation session is comprised of six series, 10, 15, 20, 25, 30, and 35 electrical stimuli (12.5–100 mA), with a 30-second interval between consecutive series [16].
In some species, however, a better response is achieved using continuous electrical stimulation. For collared peccaries (Pecari tajacu) EEJ (Table 1), the stimulatory cycle consists of 10 stimuli in each voltage, starting from 5 V, followed by voltage increases of 1 V up to 12 V, then, the voltage remains being applied during 10 minutes, continuously [17].
\n\t\t\t\tParameters\n\t\t\t | \n\t\t\t\n\t\t\t\t\n\t\t\t\t\tCollared peccary\n\t\t\t\t\n\t\t\t\t \n\t\t\t\t[18]\n\t\t\t | \n\t\t\t\n\t\t\t\t\n\t\t\t\t\tAgouti\n\t\t\t\t\n\t\t\t\t \n\t\t\t\t[14]\n\t\t\t | \n\t\t\t\n\t\t\t\t\n\t\t\t\t\tRing-tailed coati\n\t\t\t\t\n\t\t\t\t \n\t\t\t\t[8]\n\t\t\t | \n\t\t\t\n\t\t\t\t\n\t\t\t\t\tSix-banded armadillo\n\t\t\t\t\n\t\t\t\t \n\t\t\t\t[12]\n\t\t\t | \n\t\t
\n\t\t\t\tSperm concentration (x106)\n\t\t\t | \n\t\t\t765.0 ± 313.7 | \n\t\t\t357.0 ± 61.2 | \n\t\t\t197.5 ± 204.9 | \n\t\t\t450.0 ± 14.0 | \n\t\t
\n\t\t\t\tMotility (%)\n\t\t\t | \n\t\t\t86.7 ± 2.6 | \n\t\t\t80 ± 29.6 | \n\t\t\t91.3 ± 9.2 | \n\t\t\t61.0 ± 7.0 | \n\t\t
\n\t\t\t\tVigor (0-5)\n\t\t\t | \n\t\t\t4.4 ± 0.3 | \n\t\t\t3.5 ± 1.9 | \n\t\t\t4.5 ± 0.6 | \n\t\t\t2.0 ± 0.2 | \n\t\t
\n\t\t\t\tViability (%)\n\t\t\t | \n\t\t\t92.3 ± 1.6 | \n\t\t\t65.5 ± 26.1 | \n\t\t\t73.1 ± 14.5 | \n\t\t\t55.0 ± 7.0 | \n\t\t
\n\t\t\t\tMembrane functional integrity (%)\n\t\t\t | \n\t\t\t75.3 ± 2.3 | \n\t\t\t68.5 ± 17.6 | \n\t\t\t74.3 ± 12.0 | \n\t\t\t46.0 ± 6.0 | \n\t\t
\n\t\t\t\tNormal morphology (%)\n\t\t\t | \n\t\t\t83.2 ± 2.2 | \n\t\t\t83.9 ± 8.6 | \n\t\t\t81.2 ± 11.8 | \n\t\t\t86.0 ± 2.0 | \n\t\t
Characteristics of semen obtained by electroejaculation from various South American species.
Obtaining viable sperm directly from the epididymis tail is an additional option for the conservation of genetic material of South American wild animals, particularly for those accidentally killed. However, different factors such as the epididymis size and vas deferens diameter [18] as well as the possibility for contamination of samples with blood or epithelial cells are considered as limiting factors [19].
Epididymal sperm can be retrieved by flotation or retrograde flushing. In the flotation method, the epididymis is cut into small pieces in a diluent solution for the removal of sperm; this is one of the preferred techniques for small species due to the reduced size of the epididymis [20]. On the other hand, the retrograde washing is accomplished by injecting a buffered solution at the vas deferens, as it was described for the agouti [21]. In some species, such as collared peccaries [22] and yellow-toothed cavies (Galea spixii) [23], both recovery methods can be conducted with no notorious differences on sperm quality.
The research on gamete preservation needs to focus on the species-specific variations in sperm physiology, as well as the changes that gametes undergo during sperm processing [24]. The factors most often cited as crucial for success include the sperm processing and packaging, type and composition of extender, duration of equilibrium time, freezing rate, storage, and thawing rate [25].
The short-term preservation of sperm on a liquid form is rarely applied for South American species. In capuchin monkey, for instance, dilution of semen in coconut water–based extender allowed its preservation for 24 hours at 33°C [16]. Moreover, the use of a TRIS extender containing egg yolk or Aloe vera extract was efficient in preserving the collared peccary semen for 36 hours at 5°C [26].
Nowadays, intensive research focuses on the development of effective methods for sperm cryopreservation. In general, methods are adapted from the most closely related phylogenetic domestic species; for example, the domestic pig serves as an experiment model for wild pigs; however, such adaptation is not always effective. This fact is verified for six-banded armadillos that present unique semen characteristics, such as high viscosity, large sperm dimensions, and rouleaux formation, which hinders the success of freezing protocols [27].
Extenders based on TRIS plus egg yolk and glycerol were reported for collared peccary semen cryopreservation [17]. Other compounds have been described, such as the INRA® extender for tapir [13], the lactose-egg yolk extender for jaguars [28], and a combination of TES and TRIS extenders for capuchin monkey [16]. Recently, a coconut water–based extender was reported for semen cryopreservation in collared peccaries [29], agoutis [30], and squirrel monkeys (Saimiri collinsi) [31].
Although most researchers use egg yolk and glycerol as main cryoprotectant, a recent study demonstrated that the dimethyl sulfoxide (DMSO) would be more appropriate for the preservation of maned wolf semen than that of glycerol. Moreover, low-density lipoprotein [32] and Aloe vera extract [33] could effectively substitute the egg yolk for the cryopreservation of collared peccary semen.
Regarding preservation of epididymal sperm from South American wild mammals, studies are scarce. For collared peccaries [34] and cavies [35], epididymal sperm was efficiently cryopreserved using TRIS extender added to egg yolk and glycerol. In agoutis, however, epididymal sperm is better cryopreserved in coconut water–based extenders [36].
Artificial insemination (AI) is the single most important technique ever devised for genetic improvement of animals. It refers to the artificial process of sperm deposition into the female genital system, at the appropriate time, seeking the fertilization of the oocyte. The main factors that determine the fertility rates derived from AI include the individual fertility of players, the way in which sperm is collected and manipulated, the ability of the inseminator, the female management (the time of insemination), the type of sperm used (fresh, chilled, or frozen), the insemination dose, and the site for the semen deposition [37].
In spite of AI popularity among domestic animals breeders, its use remains limited in South American wild animals. Initial studies include the successful production of offspring derived from laparoscopic intrauterine AI in ocelots. One female was inseminated with 7.5 × 106 frozen-thawed spermatozoa after receiving 500 IU equine chorionic gonadotropin (eCG) and 225 IU human chorionic gonadotropin (hCG), giving birth to a male offspring after 78 days of gestation [38]. Moreover, deposition of fresh or cryopreserved semen, as well as cryopreserved epididymal sperm, into the cervix of marmoset (Callithrix jacchus) resulted in pregnancy and the production of offspring [39].
The preservation of testicular tissue for prolonged periods is one big challenge in the field of cryobiology, because spermatogonia, Sertoli, and Leydig cells contain large amounts of water, therein increasing the risk for intracytoplasmic crystal formation [40]. If the testicular tissue has preserved active spermatogenesis, it can be used for the extraction of sperm or elongated spermatids that could be used for oocyte fertilization using intracytoplasmic sperm injection (ICSI) [31]. Moreover, testicle freezing can be used in cases where the animal dies suddenly [41].
Although this is a promising area, studies on the collection of sperm or even on the preservation of testicular tissues are very scarce. In South American individuals, the unique published results can be found in collared peccaries, in which the first successful xenotransplantation of fresh testicular tissue was also documented [42]. In addition, its testicular tissue was efficiently vitrified using ethylene glycol (EG) as cryoprotectant at 3.0 or 6.0 M [43].
For wild females exhibiting irregular or regular cycles of sexual activity, estrus synchronization support assisted breeding procedures particularly to maximize the chances of conception and the use of fresh or cryopreserved semen [44]. The administration of exogenous hormones, whether or not in association, can artificially synchronize the estrous and ovulation of wild females, altering its endogenous endocrine environment [45]. Research has dramatically increased the number of synchronization protocols for wild animals, being the prostaglandins, progestins, or gonadotropins the most available, each with specific functions and peculiar mechanism of action.
One of the most ancient methods for estrus synchronization is the use of a luteolytic agent such as the prostaglandin F2α (PGF2α) or its analogues, which provokes the corpus luteum degeneration [46]. Another approach is by using gonadotropin-releasing hormone (GnRH), which represents the final common pathway where by internal and external relevant stimuli converge to control reproduction. GnRH neurons, thus, stimulate pituitary gonadotropin secretion to appropriately regulate gametogenesis and sex steroid secretion [47]. On the other hand, progestins inhibit the synthesis and secretion of GnRH that subsequently inhibits follicular growth and development as well as the ovulation [48]. Additionally, gonadotropin-based protocols include the use of equine and human chorionic gonadotropins (eCG and hCG, respectively), as well as the application of follicle stimulating hormone (FSH) and luteinizing hormone (LH), as a synthetic or extract form [49]. The action of eCG is similar to that of FSH at stimulating the ovary follicles to produce mature oocyte, thus promoting the outward signs of estrus [50]. In contrast, the hCG action is similar to that of LH, causing the release of mature oocyte at ovulation, and promoting corpora lutea formation [51]. Currently, the majority of studies focus on the use of hormonal associations. This is acceptable because the results of studies have shown that such protocols increased rate, occurrence, and ovulation speed, therefore increasing the fertility rate [52].
Various attempts have been made to establish effective protocols for estrous control in South American wild felines, mainly due to the variety of manifestations of reproductive cycles. For example, the margay present spontaneous ovulations during normal estrous cycles, but ocelot and tigrina are induced ovulators. Luteal control in feline is not a feasible option, due to several factors: (1) although some individuals ovulate spontaneously at unpredictable intervals, many felids are obligated to induce ovulations and (2) the diestrus corpus luteum, when present, is refractory to prostaglandins and dopamine agonists up to day 40 postovulation [50].
The use of porcine FSH (pFSH) with minimal LH activity is reported for inducing follicular growth in wild felines [53]. This protocol was effectively applied for pumas (Puma concolor) [54], jaguars (Panthera onca) [55, 56], and jaguarundis (Puma yagouaroundi) [49]. Recently, the ability of an oral progestin, altrenogest (0.192 mg/kg, 14 days), to suppress the ovarian follicular activity in tigrina was demonstrated. This protocol was also effective in reducing hyperstimulation and hyperestrogenism after hCG and eCG administration. However, the authors affirmed that not all females responded uniformly [45]. In fact, studies with both conventional gonadotropins showed diversity of response in different feline species. For example, the ovarian response to a combined treatment with eCG injection followed 80-84 hours later by a hCG administration resulted in a conception rate lower than 30% in ocelots (400 IU eCG and 200 IU hCG) [38] and tigrina (200 IU eCG and 150 IU hCG) [51]. Exogenous gonadotropins are immunologically complex foreign peptides, and thereby, the development of antibodies against gonadotropin is expected sequelae in wild feline submitted to short-term repeated treatments [57]. For this motive, the isolation, characterization, and production of recombinant gonadotropins has been suggested to avoid these complications. On the other hand, it is important to remember that felid species present a large variation in body sizes, a factor that directly influences the synchronization protocols, as well as the individual ovarian response to gonadotropin stimulation [58].
A considerable effort has been made to develop a method to induce estrus and ovulation in the maned wolf, the biggest South American canids. An implant of deslorelin (2.1 mg), a GnRH analogue, was 100% effective for induction of ovary activity, with ovulation occurring between days 9 and 16 (mean: 12.5 ± 1.4 days) after implant when paired with a male, a physiological condition required for the effective ovulation. Alternatively, a single injection of equine recombinant LH (reLH), associated with the deslorelin removal effectively induced ovulation in unpaired females, without apparent adverse impact on fertility in subsequent breeding seasons [59]. Regarding the coati, a carnivore from the Procyonidae family, the unique attempt of estrous control was made by using melengestrol acetate, a progestin, but treatments induced purulent vaginal discharge and uterine adenocarcinoma, and consequently the authors recommend caution when using this drug [60].
In ungulates, as the brown brocket deer (Mazama gouazoubira), a monovulatory nonseasonal breeder, estrous synchronization can be achieved using an intravaginal progesterone device (CIDR®) for 8 days, associated with an injection of 265 µg cloprostenol at the removal of the device [61]. In collared peccaries, on the other hand, the use of two injections of 60 µg cloprostenol administered at a 9-day interval was effective for estrous synchronization [62].
In primates, it is known that PGF2α can act on luteal cells to inhibit the luteotrophic actions of LH or hCG in vitro [63]. In this context, the administration of cloprostenol has a marked and rapid luteolytic action (0.5–0.8 µg) in the marmoset monkey [64]. In this species, the daily use of recombinant human FSH (rehFSH; 50 IU) during the first 6 days of the follicular phase resulted in superovulation [65]. Moreover, when associated, the protocol using rehFSH 25 IU/hCG 500 UI proved to be efficient to collect oocytes [66].
Despite the difficulties in using a great number of individuals for experimentation, there is notably a need for the development of estrous synchronization protocols for South American wild mammals, especially focused on dosage and efficiency of drugs.
As with other ARTs, the main limitation for wild species is the difficulty in obtaining viable oocytes, being the recovery of preovulatory oocytes for in vitro culture (IVC), an available alternative [67]. The possibility to store frozen female gametes as ovary tissue fragments, isolated follicles, or either mature or immature oocytes represents an attractive alternative to cryopreservation [68]; however, the technique is not yet well-established, presenting several challenges that need to be addressed in order to be routinely used.
The first step in the manipulation of female gametes is to obtain viable oocytes; however, such procedures are still uncommon in South American wild mammals and are most frequently conducted in Neotropical primates. Oocyte can be collected in vivo, or postmortem.
The recovery of oocytes by mechanical preantral follicle (PF) isolation in ovariectomy specimens [69], the collection of ovarian biopsies by exploratory laparoscopy [70], and the puncture of antral follicles by laparotomy [71] have been described studies in capuchin monkeys (Sapajus apella). In marmoset monkeys, oocyte recovery was achieved through follicle aspiration after laparotomy [72], laparoscopy [65], or uni- or bilateral ovariectomy [73]. The latter was also the method described in squirrel monkeys (Saimiri boliviensis boliviensis) [74] for oocytes collected by ultrasound-guided puncture, after monitorization of the follicular development, in superstimulated females [75]. Finally, in Saimiri sciureus, follicle aspiration was conducted after laparoscopy [76] or laparotomy [77].
Regarding other South American species, in particular, wild felids, ovary collection is often performed immediately after the animal death, as it was reported for Geoffroyi and tigrina cats [78]. Also in puma and jaguar, isolation of small PFs (40-90 µm) was performed after animal death [79]. The oocyte aspiration by laparoscopy was also described for tigrina and ocelot [51], puma [80], and jaguar [55]. In vicunas (Vicugna vicugna), a South American wild camelid, oocytes also were recovered after follicle aspiration [81].
The rescue and manipulation of matured oocytes represent an important genetic source from wild species; nevertheless, it faces many obstacles, particularly the limited knowledge on the species reproductive physiology [82]. Thus, the use of immature oocytes from antral follicles or PFs allows the recovery of gametes from prepubertal, pregnant, old, or even dead animals [83, 84] The characteristics of the ovarian follicles population in various South American wild mammals are described in Table 2 and illustrated in Figure 2.
Specie | \n\t\t\tPreantral follicles population | \n\t\t\tPreantral follicles category | \n\t\t\t\n\t\t | |||||||||||
\n\t\t\t | Primordial | \n\t\t\tPrimary | \n\t\t\tSecondary | \n\t\t\tAuthors | \n\t\t||||||||||
\n\t\t\t | Right ovary | \n\t\t\tLeft ovary | \n\t\t\tTotal | \n\t\t\tTotal number | \n\t\t\t% | \n\t\t\tDiameter (µm) | \n\t\t\tTotal number | \n\t\t\t% | \n\t\t\tDiameter (µm) | \n\t\t\tTotal number | \n\t\t\t% | \n\t\t\tDiameter (µm) | \n\t\t\t\n\t\t | |
\n\t\t\t\tSapajus apella\n\t\t\t\t \n\t\t\t | \n\t\t\t56.9 ± 21.9 | \n\t\t\t49.1 ± 26.9 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t30.0 ± 4.3 | \n\t\t\t22.1± 0.5 | \n\t\t\t- | \n\t\t\t6.0 ± 1.0 | \n\t\t\t27.3 ± 0.5 | \n\t\t\t- | \n\t\t\t4.0 ± 0.7 | \n\t\t\t61.2 ± 4.0 | \n\t\t\t[85] | \n\t\t|
\n\t\t\t\tSaimiri sciureus\n\t\t\t\t \n\t\t\t | \n\t\t\t- | \n\t\t\t- | \n\t\t\t- | \n\t\t\t915.0 ± 78.8 | \n\t\t\t73.3 ±1.3 | \n\t\t\t20.5 ± 0.8 | \n\t\t\t230.5 ± 20.8 | \n\t\t\t18.6 ± 0.7 | \n\t\t\t33.7 ± 2.4 | \n\t\t\t115.9 ± 15.7 | \n\t\t\t8.1 ± 0.9 | \n\t\t\t512.5 ± 67.1 | \n\t\t\t[86] | \n\t\t|
\n\t\t\t\tDasyprocta leporina\n\t\t\t\t \n\t\t\t | \n\t\t\t4419.8 ± 532.3 | \n\t\t\t5397.5 ± 574.9 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t86.6 | \n\t\t\t18.6 ± 3.4 | \n\t\t\t- | \n\t\t\t13.0 | \n\t\t\t23.8 ± 5.7 | \n\t\t\t- | \n\t\t\t0.4 | \n\t\t\t88.6 ± 17.6 | \n\t\t\t[87] | \n\t\t|
\n\t\t\t\tGalea spixii\n\t\t\t\t \n\t\t\t | \n\t\t\t220.8 ± 175.1 | \n\t\t\t195.2 ± 182.8 | \n\t\t\t416.0 ± 342.8 | \n\t\t\t- | \n\t\t\t32.2 | \n\t\t\t16.6 ± 0.3 | \n\t\t\t- | \n\t\t\t63.7 | \n\t\t\t28.3 ± 2.1 | \n\t\t\t- | \n\t\t\t4.1 | \n\t\t\t123.7 ± 18.3 | \n\t\t\t[88] | \n\t\t|
\n\t\t\t\tPecari tajacu\n\t\t\t | \n\t\t\t- | \n\t\t\t- | \n\t\t\t33273.5 ± 579.0 (per ovary) | \n\t\t\t30 466.6 ± 5194.8 | \n\t\t\t91.6 | \n\t\t\t31.8 ± 1.1 | \n\t\t\t2 093.9 ± 595.0 | \n\t\t\t6.3 | \n\t\t\t40.9 ± 2.1 | \n\t\t\t712.9 ± 95.4 | \n\t\t\t2.2 | \n\t\t\t196.2 ± 17.1 | \n\t\t\t[89] | \n\t\t
Characterization of ovarian follicles population from different South American wild species
Photomicrograph of the ovary cortex. (A-C) Preantral follicles from Pecari tajacu and (D-F) Dasyprocta leporina. Primordial (A,D), primary (B,E), and secondary (C,F) follicles.
The IVC and in vitro maturation (IVM) of oocytes has highlighted some of the mechanisms underlying the folliculogenesis in particular wild species, particularly among the South American wild mammals, offering new opportunities for the use of many other ARTs [85]. Oocyte IVM is a technique that provides material for the study of the final steps of oocyte meiosis. Nevertheless, the practical application of this procedure remains relatively inefficient, and the embryo production rate is much lower compared with in vivo matured oocytes [73]. Some of the researches that have been developed using IVC and IVM are revised below.
The competence of oocytes retrieved from antral follicles in capuchin monkeys was achieved after 36 hours of IVM, the highest maturation rate occurring in oocytes collected from dominant follicles [71]. It has also demonstrated that the IVC of ovarian cortical strips in TCM199 supplemented of β-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) promoted a follicular viability similar to that of controls (89.3%) for this species, while it also increases the rate of secondary follicle formation (44.9%) [70].
The oocyte meiotic competence and the cumulus cell function were positively associated with the follicle size in marmoset monkeys [86], as it was demonstrated in IVM studies. It was shown that chromosome quality is crucial for cytoskeletal organization allowing a correct meiosis in IVC and IVM of the immature oocyte [73]. Moreover, abnormal spindle formation was observed in oocytes derived from small antral follicles failing to complete meiosis, contrasting almost 90% matured oocytes, surrounded by expanded cumulus cells at the time of isolation [66]. For this species, an alternative two-step culture system consisting in the culture of oocytes within stromal tissue fragments for 2 days has been described. Afterwards, follicles were mechanically isolated and transferred to a culture system where they grew for up to further 12 days. This process produces full-sized matured oocytes from primary and early secondary follicles [87].
An immunohistochemical assay conducted in squirrel monkey ovaries during IVC demonstrated that growth differentiation factor 9 (GDF-9) and c-Kit protein were detectable in oocyte cytoplasm from primordial to secondary follicles, whereas the Kit Ligand expression was observed in oocytes and granulosa cells from primordial to secondary follicles. On the other hand, the anti-Müllerian hormone was expressed in primary and secondary follicles, but not in the primordial ones [88]. In a different species from the same gender, the S. boliviensis, IVM of follicles obtained by laparoscopy requested the use of a medium containing high-energy source [89]. Moreover, oocyte IVM was also described for S. scierus [90].
Regarding South American wild felids, the yield of the IMV technique varies greatly among species. Immature oocytes were recovered from Geoffroyi cat, jaguar, and puma after the animal death and further matured in vitro. In Geoffroyi cat, from a total of 45 oocytes retrieved, 23% succeed maturation, similarly to that described for jaguar, from whose oocytes (n=21) only one-third advanced to metaphase II during IVM. In the puma, more than 100 oocytes were recovered from 8 females, from which 43.8% of the oocytes matured in vitro [91]. IVM has been previously described in this species after oocyte recovery using laparoscopy and transabdominal aspiration [92]. In a recent study, 42 mature oocytes were collected by laparoscopic ovum pick up from pumas, after the administration of eCG (750 UI) with hCG (500 UI). In this species, oocytes were also matured in vitro using TCM199 supplemented with LH, FSH, and 17β-estradiol [80].
Studies on ovarian gene expression have been conducted to better understand folliculogenesis in plains viscacha (Lagostomus maximus), an animal showing the highest ovulation rate among all mammals, releasing between 400 and 800 oocytes per estrous cycle [93]. The expression of germ cell–specific VASA protein, apoptotic proteins BCL2 and BAX, as well as DNA fragmentation revealed an unrestricted proliferation of germ cells in this species, without apoptosis-driven elimination, contrary to what is normally found in other mammals [94, 95].
In ungulates, IVC of collared peccaries ovarian tissue reveals that more than 50% follicles remain morphologically intact when the TCM199 medium was used whether supplemented or not with FSH. Moreover, the activation of collared peccaries PFs was stimulated by the addition of FSH to the medium during IVC, the proportion of growing PFs increased from only 31.2 ± 0.7% in the control group to more than 90% after a 7 day in TCM199+FSH [96].
The conditions required for the complete in vitro development of oocytes remain incompletely established in wild species; therefore, cryopreservation emerges as a promising tool for female germplasm conservation, allowing its future use in ART. It is possible to store ovarian tissue, isolated follicles, and mature or immature oocytes, for which purpose two methods are routinely used: the slow-freezing procedure and vitrification. The value of vitrification has been highlighted to avoid damages caused by conventional freezing protocols, such as the risk of ice crystal formation. It is considered a cheap method that can be performed under field conditions.
Another major factor on the success of cryopreservation of female gametes is the cryoprotectant used. In marmoset monkeys, DMSO was advantageously used as a cryoprotectant for slow freezing of ovarian tissue. It provided a higher percentage of morphologically normal primordial (26.2 ± 2.5%) and primary follicles (28.1 ± 5.4%) compared with propanediol (PROH) (12.2 ± 3.0 and 5.4 ± 2.1%, respectively) [97]. Furthermore, the development up to secondary PFs [98] and the formation of antrum after xenografting cryopreserved tissue in this species were also described [99].
Regarding South American wild felids, short-term IVC of ovarian cortex slices collected from Geoffroyi cat was performed to assess the success of the slow freezing protocol, using EG and sucrose. The results showed an increase in the number of viable PFs in frozen–thawed samples after 7 days of IVC (48%) compared with fresh cultured pieces (31%) obtained in the same sampling day. In tigrina cats, the percentage of primordial follicles after freezing and culture (44%) indicated a normal population of viable follicles [78].
Regarding Neotropical rodents, the conventional freezing was adopted to preserve ovarian fragments from agoutis. After freezing, no differences were found among groups using different cryoprotectants: DMSO (60.6 ± 3.6%), EG (64.0 ± 11.9%), or PROH (62.0 ± 6.9%). However, only follicles cryopreserved with PROH presented a normal ultrastructure, similar to that of the control group [100]. A reduction on the morphologically normal PFs (69.5%) compared with the control group (91.2%) was also observed, after solid surface vitrification of ovarian tissue from yellow-toothed cavies. The preservation of oocytes and granulosa cell membranes and the morphological aspect of follicles were acceptable, and the transmission electronic microscopy showed that the presence of vacuoles in the oocyte and granulosa cells cytoplasm and turgid mitochondria remained the main alteration observed in some vitrified follicles [101].
In collared peccaries, the refrigeration of ovaries for up to 36 hours allowed supporting the morphological integrity and viability of PFs. Further, it was demonstrated that powdered coconut water media (66.7%) was more effective than the phosphate buffered saline (PBS, 49.4%) to preserve the morphological integrity after 36 hours storage, although without statistical differences on respect to the follicular viability [102]. Moreover, the solid surface vitrification of ovarian tissue using EG, DMSO, or dimethylformamide (DMF), at 3.0 or 6.0 M, preserved the morphological integrity of more than 70% PFs using any of these cryoprotectants [103].
ARTs using in vivo embryo production by applying hormonal superovulation protocols, as well as those obtaining in vitro blastocyst through in vitro fertilization (IVF) and ICSI, allowed to establish the adequate conditions for embryonic development in different species. Furthermore, ARTs associated with embryo cryopreservation foster the preservation of genetic material, contributing to conservation of biodiversity. In general, ARTs applied to wild mammals in South America are established in accordance with procedures used in domestic mammals, despite that some wild species may have complex reproductive characteristics, compromising the rapid progress of ARTs in particular species.
Soon after the recognition of the gametes and embryo physiology, application of ARTs in each new species begins with in vivo embryo production, defined as the selection of female donors, AI or natural mating, embryo collection, cryopreservation, or direct transfer into synchronized recipients. Inevitably, the number of individuals is a crucial point in research, and individual variations nurture the development of in vitro ARTs.
In general, superovulatory protocols consist of a single dose of eCG and/or multiple injections of FSH [104]. The status of follicular development at the moment of superovulation, as well as the stress induced by intense manipulation during ARTs, is the main factor ascribed for the variability on response [105]. In an attempt to provide efficient superovulation protocols for embryo collection, different studies have been developed in various wild mammals. An interesting example corresponds to brown brocket deer [106], in which eCG induced a good response to superovulatory protocol, promoting the formation of functional corpora lutea (7.0 ± 1.8) although 66.7% (4/6) of the females showed premature luteal regression. In comparison, FSH administration resulted in a low formation of corpora lutea (2.6 ± 0.8) and lower proportion premature luteal regression (33.3%; 2/6).
There are few studies on in vivo embryo production protocols in wild camelids. In vicunas, two studies [107, 108] proposed ovarian superstimulatory protocols using eCG with or without CIDR® during 5 days; unfortunately, the results involved only data for follicular development, with no information on embryo recovered or transferred.
The IVF is still the main in vitro technique for the reproduction of wild mammals, although this technique demands a wide quantity of viable oocytes to be fertilized. Briefly, IVF steps involve the oocyte recovery, the in vivo or IVM and selection of oocytes, the IVF using capacitated spermatozoa, and the preimplantation embryonic development. The aim is to develop high-quality embryos and obtain normal pregnancies, resulting in the birth of healthy offspring. In this sense, Table 3 summarizes the results of in vitro ARTs applied in some South American wild mammals. In general, the protocols used for domestic animals were gradually adjusted to wild species.
Although IVF embryos have been produced from capuchin monkeys [109], marmoset monkeys [66, 110], pumas [92], and tigrinas [51], modifications are needed to increase the efficiency of protocols. Currently, in vitro embryo production depends primarily on two major products: meiotic viable oocytes (meiotic competence) and capacitated viable spermatozoa (sperm competence). Domingues et al. [71] reported the viability of in vitro matured oocytes collected from nonstimulated capuchin monkeys. Lima et al. [109] established 40 hours as the optimal IVM length and showed the positive effect of FSH/LH (0.5 µg/mL/50.0 µg/mL) on IVF of oocytes collected also in nonstimulated females. In addition, spermatozoa obtained from semen diluted in coconut water were able to fertilize oocytes. However, the establishment of experimental conditions is still a requirement for this species.
Another interesting example of the use of IVF in wild felids [111] is that of jaguars; a stimulation protocol using FSH/LH resulted in approximately 25 follicles/female, with more than 80% of high-quality oocytes but the fertilization rates were fairly low (<25%) [55]. Contrastingly, ocelot and tigrina each treated with eCG/hCG produced an average of approximately 10 follicles with ~7–9 high-quality oocytes/female that resulted in 76 ocelot and 52 tigrina embryos [51].
Species | \n\t\t\tOocyte source | \n\t\t\tMaturation type | \n\t\t\tSpermatozoa | \n\t\t\tART applied | \n\t\t\tEmbryo production | \n\t\t\tAuthors | \n\t\t|||||
Collection/selection | \n\t\t\tMotility (%) | \n\t\t\tMediaa\n\t\t\t | \n\t\t\tCleavage rateb\n\t\t\t | \n\t\t\tBlastocyst ratec\n\t\t\t | \n\t\t\tStage reached | \n\t\t\tEmbryo destination | \n\t\t|||||
\n\t\t\t\tSapajus apela\n\t\t\t | \n\t\t\tWithout stimulation | \n\t\t\t\n\t\t\t\tIn vitro\n\t\t\t | \n\t\t\tEEJ, cooled to 4°C/ Swim up | \n\t\t\t~80 | \n\t\t\tIVF | \n\t\t\tSOF | \n\t\t\t20 | \n\t\t\t--- | \n\t\t\t4 cells | \n\t\t\tCell staining | \n\t\t\t[114] | \n\t\t
\n\t\t\t\tCallithrix jacchus\n\t\t\t | \n\t\t\tHormonal stimulation | \n\t\t\t\n\t\t\t\tIn vivo\n\t\t\t | \n\t\t\tFertiCare vibrator, artificial vagina/ Pure gradient | \n\t\t\t"/>70 | \n\t\t\tIVF/ICSI | \n\t\t\tG1.2, G2.2 | \n\t\t\t44.7/94.2 | \n\t\t\t46.7/0.0d\n\t\t\t | \n\t\t\tBlastocyst | \n\t\t\tCell staining | \n\t\t\t[66] | \n\t\t
\n\t\t\t\tCallithrix jacchus\n\t\t\t | \n\t\t\tHormonal stimulation | \n\t\t\t\n\t\t\t\tIn vitro\n\t\t\t | \n\t\t\tFertiCare vibrator, artificial vagina/ Swim up | \n\t\t\tNI | \n\t\t\tIVF/ICSI | \n\t\t\tISM1, ISM2 | \n\t\t\t93.2/97.6 | \n\t\t\t39.2/35.4 | \n\t\t\tBlastocyst | \n\t\t\tEmbryo transfer | \n\t\t\t[115] | \n\t\t
\n\t\t\t\tFelis concolor\n\t\t\t | \n\t\t\tHormonal stimulation | \n\t\t\t\n\t\t\t\tIn vivo\n\t\t\t | \n\t\t\tEEJ/ Swim up | \n\t\t\t40-50 | \n\t\t\tIVF | \n\t\t\tmKRB | \n\t\t\t--- | \n\t\t\t--- | \n\t\t\t1 cell | \n\t\t\tNI | \n\t\t\t[97] | \n\t\t
\n\t\t\t\tPecari Tajacu\n\t\t\t | \n\t\t\tWithout stimulation | \n\t\t\t\n\t\t\t\tIn vitro\n\t\t\t | \n\t\t\tTestis cell suspension xenografts/--- | \n\t\t\tNI | \n\t\t\tICSI | \n\t\t\tNI | \n\t\t\t37.5 | \n\t\t\t--- | \n\t\t\t4 cells | \n\t\t\tXenograft assay | \n\t\t\t[42] | \n\t\t
Results of IVF and ICSI techniques applied in some South American wild mammals.
Abbreviations: EEJ, electroejaculation; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; ARTs, Assisted reproductive technique; NI, not informed.
aMore information about the composition of the media see reference.
bCleavage rate represents the number of cleaved oocytes in relation to the number of oocytes entering to maturation in D2.
cBlastocyst rate represents the number of blastocyst in relation to the number of zygotes.
dBlastocyst rate represents the number of blastocyst in relation to the number of cleaved embryos.
Alternatively, ICSI offers a precise control of sperm competence for in vitro procedures. ICSI was first used in marmoset monkeys in 2007, by Grupen et al. [66], using in vivo matured oocytes. Still, no blastocyst was produced by ICSI, despite that 47% of blastocysts were achieved after IVF. In 2014 [110], ICSI embryos derived from in vitro matured marmoset oocytes developed into neonates, through the use of two strategies: the evaluation of the most suitable timing for ICSI and establishing the in vitro and in vivo embryo developmental potential. In this study, the blastocyst rate tended to be low when ICSI was performed 1–2 hours after the extrusion of the first polar body. For in vitro developmental competence, a greater fertilization rate was observed in ICSI embryos (93.2%) compared with IVF embryos (82.2%). However, no differences in developmental rate (blastocyst/fertilized oocytes) were observed between ICSI (35.4%) and IVF (39.2%). In addition, for in vivo development of ICSI embryos, an offspring rate of 28.6% (6/21) and 2.7% (1/37) was obtained after the transference of 6 to 8 cell embryos and blastocysts, respectively.
In collared peccary [42], ICSI was used to evaluate the quality of sperm cells derived from testis cell suspension xenografts. The collared peccary presents an interesting Leydig cell cytoarchitecture, and therefore, it represents an interesting mammalian model for investigating cellular roles in male gonads. In the study, the sperm recovered from the xenografts originated 75.0% (6/8) of two-cell embryos and 37.5% (3/8) of four-cell embryos produced in 24 and 48 after ICSI, respectively.
For South American wild camelids, information on oocyte recovery and maturation are only available for vicuna: using the surgical oocyte aspiration from superstimulated females, a recovery rate of 55.4% (46/83) and an IVM rate of 41% were obtained, characterized by extrusion of the first polar body and cytoplasmic maturation [81], but no information was presented on IVF or ICSI.
In recent years, advances in the embryo cryopreservation were achieved with studies in slow freezing conventional method or vitrification. Currently, embryo cryopreservation targets the establishment of a consistent vitrification protocol to be applied for the conservation of preimplantation embryos in different stages of development. Protocols developed for other species appear to be adequate for the purpose of embryo cryopreservation in wild felids. Offspring were obtained in ocelot following transfer of cryopreserved embryos [111].
In marmoset monkey, due to its small body size, the technical development of an efficient embryo transfer is being delayed. Different factors are being studied, such as nonsurgical approaches, embryo cryopreservation, use of late-stage embryos, and volume medium [112] to overcome these gaps. Using embryos in different developmental stages (10- to 16-cell, morula, and blastocysts) after vitrification in Cryotop, Ishibashi et al. [112] defended that reducing the transfer volume to 1 µL or less is essential for successful embryo transfer this species. This procedure provides pregnancy and birth rates of 80% (8/10) and 75% (9/12), while the use of larger volumes (2–3 µL) results in pregnancy and birth rates of 50% (5/10) and 27% (3/11), respectively.
Somatic cell nuclear transfer (SCNT, cloning) consists of the transfer of the nucleus of donor cells into enucleated oocytes, resulting in the production of an individual genetically identical to the nucleus donor animal. In general, SCNT is performed according to the following steps: (i) preparing cytoplast receptors from oocyte recovery, selection, and maturation (cytoplast); (ii) isolation, characterization, IVC, and cellular cycle synchronization of the somatic cells to be used as nuclear donors (karyoplast); (iii) embryonic reconstruction by nuclear transfer, fusion of karyoplast–cytoplast complex, cellular activation, and preimplantation embryo development in vitro; and (iv) transfer of embryos for previously synchronized recipients, establishment of pregnancies, fetal development, and birth of offspring. In all these steps, there are determining factors for the success of the technique, including the media composition, the donor cell type and the oocyte source, and the timing of all steps down the success of the reproductive output of viable clones [113].
The use of cloning for conservation is often questionable for the risk of reducing the genetic variability. However, it is now known that SCNT not only provides the production and multiplication of a group of individual species of interest, as well as it allows additional studies in the field of reprogramming and cryopreservation of somatic tissue and cells, providing a larger scene in the reproduction of these species. Up to now, there are no studies on clone embryo production in South American wild mammals.
An important step in cloning is the selection of the cells donating a nucleus. In general, fibroblasts derived from skin biopsies are the cell model used. Numerous reasons justify its use for embryo reconstruction, such as the ease of obtaining and handling for the primary culture or subcultures, freezing patterns, and suitable transfection, in addition to obtaining inexhaustible quantities from a single explant. Moreover, the preservation of cells and tissues of the animal to be cloned allows the preservation of the genetic material of an individual indefinitely.
The karyoplast preparation derived from skin biopsies involve multiple steps, harvesting of the biological material, through the isolation, establishment and characterization of cell cultures, and the cryopreservation conditions of cell populations after subculture cycle. Routinely, karyoplasts may be derived from skin biopsies of adult or fetal auricular region, according to established standards of asepsis and sterilization procedures [113]. After processing tissue samples into smaller fragments, these are distributed in Dulbecco Modified Eagles Medium (DMEM) supplemented with sodium pyruvate, sodium bicarbonate, antibiotics, amphotericin B, and fetal bovine serum. The primary cultures are monitored every 24 hours to evaluate the cell growth and total medium replacement. When 70% cell confluence is purchased on the plates, the cultures are subcultured. In general, explants are cultured under these conditions for 7–14 days [114]. To confirm the cells’ characteristics, particular antibodies are used, like vimentin [113].
In native Chilean species, the establishment of donor cell was already observed in Chilean Shrew opossum (Rhyncholestes raphanurus) and chinchilla (Chinchilla lanigera) [115]. In the study, tissue fragments were subjected to culture systems and somatic cells were obtained for future cloning.
The tissue cryopreservation involves some major issues, such as the conservation of cellular functionality, extended storage of the samples, and the easiness of the procedures performed outside the laboratory [115]. Tissue sample cryopreservation of wild species is an interesting step for biodiversity conservation, when there is not an appropriate culture system.
In collared peccary [114, 116], vitrification techniques (conventional or solid-surface vitrification) may be used for cryopreservation of somatic tissue, allowing the isolation of viable cells. In this study, ear fragments (9.0 mm3) were vitrified in a solution containing DMEM plus 3.0 M DMSO, 3.0 M EG, 0.25 M sucrose, and 10% fetal calf serum. The histological analysis and IVC showed that few tissue damages were associated with solid-surface vitrification [116]. Moreover, all cells resulting in vitrified and noncryopreserved fragments showed the characteristics of suitable fibroblasts: fusiform features with oval nucleus in the center. The growth curves showed clear log and lag phases of development, with no difference between treatment and nonvitrified for population doubling time (conventional vitrification directly: 66.1 hours; solid-surface vitrification: 66.3 hours; and nonvitrified: 54.0 hours) [114].
Although some domestic mammals have already been cloned, information on cloning of South American wild animals is scarce. As with other animal species, cloning allows to know the principles of nuclear reprogramming, conservation of cells and tissues for different purposes (reproductive and therapeutic), and clones can also be used as models in several studies. Thus, the efficient production of pluripotent stem cells (iPSC) allows the generation of genetic preservation by different ways, such as gamete production and embryo complementation. Dermal fibroblasts derived from jaguars were transduced using genes encoding the human transcription factors, in an experiment that highlighted the homeobox protein NANOG as crucial to the cell reprogramming in this species, and demonstrated that the technique may represent an efficient method for iPSC production from endangered felids [117].
The ARTs have applications in different productive and scientific sectors, whether or not aiming at the conservation of species. As occurred for the domestic species, the use of ART sequence followed the order of complexity and evolution of biotechnologies. Accordingly, the routine use of AI and embryo transfer in different species of mammals is evident. Moreover, in vitro ARTs continue to grow, using a parallel between the protocols applied to domestic species and individual characteristics of each wild ones. With the progress of in vivo or in vitro ARTs, it is expected to know, to manipulate, and to conserve the genetic material, promoting the preservation of biodiversity, especially in South American wild mammals.
Keypoints
\nThe practice of physical activity, healthy eating, quitting smoking, and reduction of alcohol consumption are factors that interfere in the reproductive outcomes. Medical understanding and ability to listen to patients about their obstetric past are fundamentally important for the treatment.
\nThe genetic investigation is controversial and consists of chromosomal evaluation of the conception products and the couple’s karyotype. The goal is to identify the etiology of the loss and may be useful for future guidance of the couple. There is no consensus on performing IVF-PGT, and this option should be discussed case by case. In extreme cases IVF using donated gametes may be the last option.,
\nPatients with RPL without other risk factors for thrombosis should not be screened for inherited thrombophilias, and those with positive screening have no benefit from available treatment. The only thrombophilia that should be routinely investigated for early miscarriage is APS. The recommended treatment is the use of low-dose AAS preconception and LMWH in a prophylactic dose initiated when diagnosing pregnancy.,
\nScreening immunological factors for patients with RPL is not recommended. There is also no recommendation to use venous immunoglobulin or corticosteroids empirically. Only antinuclear antibody can be ordered for prognostic purposes, according to ESHRE.,
\nScreening for congenital uterine anomalies is part of the investigation of women with a history of RPL. Nuclear magnetic resonance is the gold standard for diagnosis. The only finding that can be surgically corrected and prognosis improved is the septate uterus.,
\nThe diagnosis of cervical incompetence is based on clinical history. The classic treatment is transvaginal cerclage between 12 and 16 weeks after first trimester morphological ultrasound., Patients with RPL should undergo through endometrial cavity evaluation. The gold standard is hysteroscopy. Although there is limited evidence linking submucosal fibroids, endometrial polyps, and synechiae with RPL, surgical correction in patients with RPL without other identifiable factors is suggested.,
\nThere are no research and treatment benefits for PCOS patients and their associated endocrine disorders. Thyroid evaluation should be performed with serum TSH and anti-TPO, and clinical hypothyroidism should be treated. For prolactin, the test is not indicated in the absence of signs of hyperprolactinemia, but if this condition is diagnosed, treatment is indicated. Vitamin D test is not routinely recommended, but the preconception counseling in women with RPL may include prophylactic vitamin D supplementation due to the high prevalence of hypovitaminosis D in this population.,
\nThe relationship of chronic endometritis with RPL is unclear. The current gold standard for the diagnosis of chronic endometritis is the pathological anatomy of immunohistochemically endometrial biopsy for the CD138 marker. A therapeutic option would be the use of doxycycline alone or in combination with other antibiotics.,
\nFor male factor, measurement of spermatic DNA fragmentation index and Kruger morphology would be indicated. The use of antioxidants is a clinical treatment that can improve DNA fragmentation. In the presence of ICSI indication associated with increased spermatic DNA fragmentation, the use of testicular, IMSI, or PICSI sperm can be considered.
\nRecurrent pregnancy loss (RPL) is defined by two or more losses with gestational age less than 20–24 weeks [1, 2]. Its prevalence varies between 0.8 and 1.4% considering only patients who have had a clinical pregnancy [2]. The pathogenesis is multifactorial, and in only 50% of the cases, the causal factor can be identified: immunological, endocrine, genetic, metabolic, and anatomical, among others [3]. The identification of etiology is not always possible, and recurrence of miscarriage seems to influence negatively the couple’s psychological profile [2]. Thus, the understanding of diagnostic methods that can identify etiological factors and treatments that can improve the outcome is fundamental for the follow-up of couples with RPL.
\nSome personal factors such as lifestyle and even environmental exposure may be associated with obstetric complications and gestational loss. Advanced maternal age is one of the best-established risk factors in the literature for RPL [2]. Approximately 50–70% of early gestational losses are associated with chromosomal abnormalities, and their incidence increases with maternal age, reaching 50% in women over 40 years [3]. The European Society of Human Reproduction and Embryology (ESHRE) recommends that women should be informed of the highest risk of miscarriage after age 40 [2].
\nObesity also has a major impact on women’s reproductive health. High body mass index (BMI) is associated with worse outcomes in infertility treatments and a higher incidence of gestational loss [4]. One study with obese women showed a higher frequency of euploid miscarriages than nonobese women (58% vs. 37%) [5]. This is probably due to the association of obesity with several endocrine disorders, such as diabetes, hypothyroidism, and polycystic ovary syndrome and possibly endometrial changes [5]. The Royal College of Obstetricians and Gynecologists (RCOG) recommends prepregnancy weight loss due to the associated increased risk of miscarriage, stillbirth, preeclampsia, diabetes, and postpartum hemorrhages [6]. The practice of regular physical activity presents improvement in the obstetric outcome; however, there are no studies investigating the impact of exercise in patients with RPL [2].
\nSmoking seems to be related to defects in trophoblastic function, thus increasing the risk of gestational loss, in addition to poor obstetric prognosis [2]. Assisted reproduction societies recommend quitting smoking because of the negative impact on the chances of a live birth [2, 3]. Several studies have shown that drinking alcohol during pregnancy also increases the risk of gestational loss [2]. Although further studies are needed to establish if there is any safe dose for drinking in pregnancy, there are recommendations for couples with RPL not to drink alcohol.
\nCaffeine abuse can also affect fertility as well as be a risk factor for gestational losses. Ingestion of high caffeine levels (500 mg per day or > 5 cups per day) is associated with decreased fertility [7]. During pregnancy, drinking between 200 and 300 mg/day (2–3 cups) may increase the risk of miscarriage [3]. Thus, it seems sensible to guide this population to reduce caffeine consumption.
\nFew studies assess environmental exposure as a risk factor for RPL, one of which suggests that exposure to heavy metals and lack of micronutrients may cause gestational loss [8]. Another study suggests that ingestion of high concentrations of organochlorine pesticides may be associated with RPL [9].
\nIt has been suggested in the past that stress could be associated with worsening the reproductive outcomes. There is a higher prevalence of depression in patients with RPL [10], but it is not known if this picture is not the cause or effect of RPL [2]. The American Society for Reproductive Medicine (ASRM) advises psychological support for these women who are more prone to feelings of grief, sadness, depression, anxiety, and guilt [3].
\nThe human conception is a vulnerable event—a large proportion of all conceptions are cytogenetically abnormal, and most of such pregnancies evolve to abortion. In couples with RPL, research can be divided into two main categories: genetic analysis of products of conception and parental genetic analysis.
\nStudies in which products of conception were analyzed showed that genetic alterations, mainly aneuploidies, contribute to a significant portion of the causes of gestational losses, accounting for 50% of recurrent losses [11]. Despite the importance of genetic alterations as causes of miscarriage, there is still no consensus as to whether routine evaluation of pregnancy tissue should be performed. ASRM does not recommend genetic evaluation of conception products [3]. ESHRE, in turn, suggests that this analysis should not be done routinely but that it may be promoted for the purpose of clarifying the etiological factor and to assist in deciding whether further investigation or treatment is needed [2]. Other studies and guidelines, however, have proposed new algorithms in which the assessment of gestational repetition losses should be initiated with chromosome testing in conception products [12].
\nNew chromosomal tests such as the chromosomal microarray analysis (CMA) have the potential to reduce costs since, in the presence of altered examination, costly and unnecessary evaluations will not be employed [13]. In addition, when a cause is identified, the tendency is to reduce the use of empirical treatments that have no scientific evidence [13]. Research has shown that in couples with previous embryonic aneuploidy, the likelihood of a child’s birth during subsequent pregnancies was higher than patients with prior normal karyotype of conception products (71% vs. 44%) [14].
\nThe suffering that the couple goes through experiencing abortion episodes without knowing the etiological factor can by itself justify the investigation of the existence of genetic alterations as a cause of the events.
\nIn about 5% of all couples suffering two or more fetal losses, one partner carries a balanced chromosomal rearrangement, which represents approximately eightfold increase compared to the general population [15]. Guiding this couple for genetic counseling is important, as the likelihood of a healthy child born will depend on the type of rearrangement found and the chromosomes involved—for example, gestational losses are more present in carriers of balanced translocations and inversions than in carriers of Robertsonian translocations [16]. Even with one spouse carrying a chromosomal rearrangement, the cumulative rate of live birth, even in natural conception, is significant—63.4% despite the increased risk for miscarriage [17].
\nAs for the existing guidelines regarding parental cytogenetic investigation, ESHRE determines that such assessment should not be performed routinely, but in specific cases after individual risk assessment [2]. ASRM, however, recommends routine parental karyotyping as information obtained may assist in counseling on the prognosis of future pregnancies, including guidance for performing preimplantation genetic testing (PGT), amniocentesis, or chorionic villus analysis [3].
\nCouples with structural cytogenetic changes have an increased number of gametes with chromosomal imbalances, so it would be expected that the implantation of embryos selected by PGT increases the rate of live births. However, in spouses carrying chromosomal rearrangement with RPL, the rate of live births, time to subsequent conception, and miscarriage rates were similar in both naturally conceived and in vitro fertilization associated with preimplantation diagnosis (IVF-PGT) [18]. Other papers showed discordant results. Similar live birth rate and time to new pregnancy were reported; however, the miscarriage rate was significantly lower in the IVF-PGT group [19]. Thus, there is no consensus showing the benefit of such strategy in this population, and no randomized controlled trials have been conducted to this date to validate possible benefits.
\nThrombophilias are inherited and/or acquired conditions that predispose individuals to thrombosis, with varied prevalence in the general population [20]. The most common hereditary thrombophilias are methylenetetrahydrofolate reductase (MTHFR) gene polymorphism 4–16%, factor V Leiden mutation 1691G → A (heterozygote, 1–15%; homozygote, <1%), prothrombin mutation 20210G → A (heterozygote, 2–5%; homozygote, <1%), antithrombin deficiency (0.02%), protein C deficiency (0.2–0.4%), protein S deficiency (0.03–0.13%) [21], and serpin gene polymorphism. On the other hand, acquired thrombophilia is mainly represented by the antiphospholipid antibody syndrome (APS) 2% [20]. Successful pregnancy requires an adequate endovascular implantation and remodeling measured by trophoblast, and these prothrombotic conditions would be the target of investigation and intervention with anticoagulant therapy to prevent miscarriage [21].
\nThe screening of inherited thrombophilias even in patients with a thrombosis context is still questioned [2]. The factor V Leiden mutation (1691G→ A) and the prothrombin mutation (20210G→ A) were related to recurrent miscarriage [22]; however, the lack of evidence that the treatment changes the gestational outcome leads to questioning the relevance of investigating such mutations. Other thrombophilias, such as protein C deficiency, protein S deficiency, and antithrombin deficiency, although associated with thromboembolic event, were not associated with RPL [2, 3, 20, 22]. MTHFR gene polymorphisms are no longer considered risk factors for thrombophilias [2].
\nThe association between RPL and inherited thrombophilias is weak or absent [2]. Thus, thrombophilic screening should be restricted to patients with family history of thrombophilias or previous thrombotic event [1, 2]. There is no recommendation to screen inherited thrombophilias in patients with RPL without other risk factors [1, 2, 21, 23]. Screening tests may be influenced by physiological/pathophysiological changes in the pregnancy-puerperal period, thrombotic event, or use of anticoagulants [21]. It should be performed within 6 weeks or more after delivery, miscarriage, or thrombotic event or early if necessary [2, 21].
\nThe use of anticoagulant therapy with low-molecular-weight heparin and/or aspirin has no benefit in preventing early (<10 weeks) or late (≥10 weeks) RPL [24]. Thus, ineffectiveness of the treatment, the risk exposure, and the increased cost do not justify treatment with anticoagulants in patients with inherited thrombophilias and RPL without other risk factors for thrombosis [2, 20].
\nAPS is indicated in patients with RPL, as well as in patients with adverse gestational outcome or episode of thrombosis without apparent cause [25]. The diagnosis of APS is based on the combination of at least one clinical criterion, which includes thrombotic events and/or gestational morbidity, and a laboratory criterion, which includes three antibodies: lupus anticoagulant, anticardiolipin, and anti-β2 glycoprotein 1 (anti-β2GP1) [25].
\nIn the cases of late gestational loss, lupus anticoagulant was more closely related to RPL than any of the other antibodies [26, 27]. Anticardiolipin (IgG and IgM) has been associated with early and late gestational loss [26, 27]. The relationship between anti-β2GP1 and late gestational loss seems to be controversial [26, 27]. ESHRE recommends for patients with two losses, consecutive or not, to conduct a research for lupus anticoagulant antibodies and anticardiolipin, and the research should consider anti-β2GP1.
\nThe use of combined therapy, low-molecular-weight heparin at prophylactic dose, and low aspirin dose (75–100 mg/day) increases the live birth rate in patients with APS and RPL from 10% to 70–80% [28]. In treatment failure, the use of heparin in therapeutic dose may be used, although there is no benefit evidence [28]. Other treatment regimens with limited evidence are the use of hydroxychloroquine or low dose of prednisolone in the first trimester [28]. The use of immunoglobulin is questioned because studies are limited and show no increase in live birth rate [28].
\nTo be successful in pregnancy, the maternal organism needs to undergo immunological changes that allow and assist in the trophoblastic invasion of the embryo. During pregnancy, the maternal immune system faces a dilemma: it needs to protect the mother against infection while accepting the semi-allogeneic fetus [29]. Leukocytes are important components of the endometrium, and their concentration increases in the middle of the secretory phase in which embryonic implantation is expected and continues to increase during early pregnancy [30]. The progesterone plays a key role in this balance by creating an appropriate environment for embryonic implantation and development [28]. This change in maternal endometrial immunology becomes essential for early pregnancy implantation and success. Changes in this phase can lead to implantation failure, miscarriage, and other unfavorable obstetric outcomes such as preeclampsia.
\nThe uterine natural killer (uNK) cells are the most commonly found leukocytes in the maternal endometrium. Two phenotypes are observed—CD56bright and CD16dim—unlike peripheral blood where CD56dim and CD16+ are the largest population [31]. There is a variability of their own concentration during the menstrual cycle. There are a significant increase of NK cells in the endometrium 6 to 7 days after the peak of luteinizing hormone (LH), which persists throughout the early pregnancy. This increase suggests an important role of these cells in embryonic implantation, but the exact function is still unknown [30].
\nThe placental formation is regulated by the interaction between the killer immunoglobulin-like receptors (KIR) and the surface human leukocyte antigens on the embryo trophoblastic cells (HLA-C). The embryo presents maternal and paternal HLA-C, and both haplotypes are presented to NK cells that, in turn, will recognize the human leukocyte antigen (HLA) foreign to their organism. There are two types of HLA-C: C1 and C2 which are a strong ligand to the receptor. On the other hand, there are two KIR haplotypes: A, which is inhibitory, and B, which is stimulating. The receptors can then be AA, AB, or BB. The presence of haplotype B confers pregnancy protection, and its absence (in the cases of KIR AA) increases the risk of gestational complications.
\nStudies have shown that when maternal KIR is homozygous for haplotype A (KIR AA), there is an increased risk of gestational complications if the embryo carries paternal HLA-C2 [32, 33]. In the future, these studies may be applicable to couples who will undergo IVF. Further studies on the subject are still needed, and these tests are not quoted to be traced by societal guidelines.
\nThe macrophages represent 20–30% of leukocytes in the maternal endometrium and are the second largest group behind only NK cells. Macrophages differ in specific phenotypes to perform different biological functions and can be divided into two subgroups: M1 and M2. M1 macrophages are pro-inflammatory and antimicrobial, whereas M2 have anti-inflammatory function [34]. For maternal and fetal tolerance to occur, more macrophages are polarized into the M2 subtype with immunosuppressive properties necessary for normal pregnancy to occur [35]. When polarization of these cells does not occur correctly favoring the M1 subgroup, improper remodeling of the arteries and trophoblastic invasion occurs, leading to a higher incidence of miscarriage, preeclampsia, and premature birth [35].
\nRegulatory T cells (Treg) are a subpopulation of T cells that play an essential role in maintaining maternal immune tolerance. These cells are activated by the presented antigens and from that moment secrete cytokines that will determine the differentiation of T cell subtypes, thus modulating the immune response. Depending on the released cytokines, T cells may differentiate into Treg cells expressing interleukin 10 and transforming growth factor β (TGFβ) responsible for immune tolerance to the conceptus or Th17 expressing interleukins 17, 21, and 22 responsible for autoimmunity and gestational loss. Treg cells will then regulate the response to foreign antigens when an aggressive response is not appropriate, having the ability to inhibit type 1 helper (Th1) cells. There is evidence in the cases of recurrent gestational loss of unknown cause to increase Th17 and to decrease Treg cells, leading to an inadequate immune response [29].
\nUterine anatomical abnormalities, both acquired and congenital, are associated with RPL. It is estimated that uterine factors may account for 10–50% of RPL [36].
\nCongenital uterine anomalies (CUA) arise from defects along any stage of the Müller duct development process during embryonic development, whether in formation, fusion, or reabsorption. The frequency of CUA has been reported between 1.8 and 37.6% in women with a history of RLP. This variation is due to the different diagnostic methods and criteria [37]. Septate uterus is the most common anomaly in patients with a history of abortion. Arched, septate, and bicornuate uterus account for up to 85% of anomalies [38].
\nIn a meta-analysis it was observed that patients with septate or bicornuate uterus had a higher rate of miscarriage in the first and second trimester than a control group [39]. In another meta-analysis, the evaluation of uterine abnormality subtypes resulting from fusion defect showed that women with unicornuate and bicornuate uterus were more likely to have first-trimester abortion compared to those with normal uterus [40].
\nASRM’s original classification system for congenital uterine anomalies has been modified and adapted and is still the most widely used today [41]. In 2012, ESHRE/ESGE published a classification system aiming to replace the subjective criteria of ASRM’s classification by the absolute morphometric criteria [42]. Based on this classification, up to 58% of women previously diagnosed with ASRM arched uterus would be reclassified as having a partial septate uterus. There would be a potential increase in the number of surgical corrections for uterine anomaly, without any evidence showing that such a practice would be beneficial [43]. Therefore, caution is needed in using this new classification until further prospective, randomized, controlled, long-term studies are available to associate the severity of uterine cavity distortion with reproductive results.
\nGiven the suspicion, it is necessary to use diagnostic methods that can clearly visualize the external contour of the uterus and endometrial cavity. Both 3D ultrasound with inversion mode (3D US) and magnetic resonance imaging (MRI) can be used for this purpose, with good correlation between them [44]. The disadvantages of MRI are that it is a more expensive and less available method than ultrasound.
\nIn a comparative study of different diagnostic modalities, higher accuracy of 3D hysterosonography compared with 3D US and 2D hysterosonography was observed, although the differences between these imaging techniques did not reach statistical significance in the diagnosis of arched, bicornuate, and septate uterus [45].
\nThe uterine septum is the most common abnormality related to RPL [36] and the only remediable one. Despite the lack of randomized and controlled prospective studies comparing surgery to expectant treatment, limited studies indicate that hysteroscopy septal resection is associated with a reduction in subsequent abortion rates and an improvement in live birth rates in patients with RPL [41]. After hysteroscopic resection of the septum, an interval of at least 2 months should be expected for complete healing of the endometrial cavity before a new pregnancy [41].
\nIn general, CUA may be associated with renal abnormalities in approximately 11–30% of individuals [41]; for this reason there is a need for urinary tract investigation in these cases.
\nCervical incompetence (CI) is the inability of the cervix to keep the intrauterine fetus in the absence of uterine contractions or labor (painless cervical dilatation) due to a functional or structural defect. It is a recognized cause of RPL in the second trimester, but the true incidence is unknown, as the diagnosis is essentially clinical [2].
\nThe CI can be congenital or acquired. The most common congenital cause is a defect in the embryological development of the Müllerian ducts. The most common acquired cause is cervical trauma, such as cervical lacerations during childbirth, cervical conization, or forced cervical dilation during uterine procedures [46].
\nThe diagnosis is usually based on a history of miscarriage in the second trimester, preceded by spontaneous rupture of membranes or painless cervical dilation. There are currently no objective tests capable of identifying women with cervical weakness in the nonpregnant state [2].
\nTransvaginal ultrasound may be used in at-risk patients during pregnancy. CI might be suspected when there is a short cervical length, less than or equal to 25 mm, or funneling, protrusion of the membrane into a dilated internal orifice but with closed external orifice [46].
\nMany surgical and nonsurgical modalities have been proposed to treat cervical incompetence. Among nonsurgical activities, restriction of activities and bed rest were not effective in the treatment of cervical incompetence. Its isolated use is discouraged. The use of vaginal pessary is another option, but the evidence is still limited. Surgical approaches include transvaginal and transabdominal cervical cerclage [46].
\nAcquired anatomical factors commonly associated with RPL include uterine fibroids, endometrial polyps, and uterine synechiae. They usually develop after puberty due to physical or hormonal stimuli and are present in about 12% of patients with RPL [47].
\nFibroid is reported in 8,2% of women with RPL [48]. Submucosal fibroids deform the endometrial cavity, thus affecting implantation and embryonic development [47]. Hysteroscopy is considered the gold standard for the diagnosis of submucosal fibroids, but this pathology can be identified through other imaging exams, such as ultrasound mapping [2]. The evaluation of the uterine cavity is strongly recommended for all women with RPL, since the removal of submucosal fibroids in infertile patients seems to reduce the chance of miscarriage [2, 49]. Regarding fibroids that do not distort the uterine cavity, there is no evidence indicating that myomectomy may reduce the chances of an abortion [2, 49].
\nThere seems to be a higher prevalence of endometrial polyps in women with gestational loss (2.4%), but with no well-defined clinical importance [2, 47]. Hysteroscopy is considered the gold standard exam for the diagnosis and treatment of endometrial polyps but can also be identified through other imaging exams, such as ultrasound with color Doppler [2]. Although there is no evidence of the benefit of polypectomy in women with RPL, hysteroscopic removal should be considered when the polyp is larger than 1 cm when no other known etiology is found [2, 47]. ASRM reports that research for uterine polyps in women with gestational loss is controversial as there is no conclusive evidence that surgical treatment reduces the risk of gestational loss [49].
\nThe prevalence of uterine synechiae ranges from 0.5 to 28% in patients with RPL [47]. Women with RPL are more likely to have uterine synechiae as they often undergo curettage or manual vacuum aspiration. The probable pathophysiology of abortion occurs due to a reduction in the amount of functional endometrium which may interfere with the invasion and normal development of the placenta [47]. The gold standard exam for the diagnosis of synechiae is hysteroscopy and should be the exam of choice in the cases of suspicion [2]. ESHRE concludes that there is insufficient evidence to recommend adhesiolysis in women with RPL as there are only small observational studies. ESHRE reinforces that treatment should focus on preventing recurrence of adhesions [2, 3]. However, ASRM points out that surgical correction of significant uterine cavity defects should be considered [3]. Nonsurgical experimental techniques for the treatment of uterine synechiae and endometrial fibrosis, such as stem cell therapy, should be further studied before being indicated in clinical practice [2].
\nHormones play a key role in placentation, and their changes may result in the risk of miscarriage [2].
\nIt is a condition of insufficient exposure to progesterone to maintain a secretory endometrium that will lead to normal embryo implantation and growth [50]. The diagnostic criteria for luteal insufficiency are not well established which makes it difficult to conduct studies that can demonstrate the causal link between luteal phase insufficiency and RPL. Thus, luteal phase failure testing is not recommended for patients with RPL [2, 3]. The use of progesterone or human chorionic gonadotropin (hCG) for its treatment is divergent in the literature [2, 3].
\nStudies relating subclinical hypothyroidism, defined as thyroid-stimulating hormone (TSH) > 2.5 mU/L and normal free thyroxine, and increased risk of RPL, have low levels of evidence [2]. The anti-thyroid peroxidase antibodies’ (anti-TPO) presence in patients with RPL, even euthyroid, is an important gestational prognostic factor [51]. Thus, a TSH and anti-TPO dosage is recommended for women with RPL. And, in detecting abnormal levels of the above exams, it recommends that T4 levels should be evaluated [2].
\nPatients with clinical hypothyroidism should be treated with levothyroxine [2, 3]. In women with RPL and subclinical hypothyroidism, the benefit of treatment should be evaluated as the evidences are conflicting [2, 3]. In addition, euthyroid women with positive anti-TPO should not be treated with levothyroxine [2, 52].
\nSeveral abnormalities observed in patients with polycystic ovary syndrome (PCOS) have been independently associated with RPL, including insulin resistance, hyperinsulinemia, hyperandrogenemia, hyperprolactinemia, and obesity.
\nThere is a higher prevalence of insulin resistance among women with RPL than controls [53]. However, no study has confirmed the cause-effect relationship between insulin resistance and RPL. Thus, there is insufficient evidence to recommend assessment of PCOS, fast insulin and fast glucose, and insulin and glycemia nor the use of metformin in pregnancy to prevent gestational loss in women with RPL and defects in glucose metabolism [2].
\nThe presence of an independent link between hyperandrogenemia and RPL remains controversial. Therefore, researching androgen levels is not recommended in women with RPL [2].
\nMost studies fail to establish a direct link between RPL and serum prolactin concentration. Thus, prolactin test is not routinely recommended in the absence of clinical signs of hyperprolactinemia [2]. But if hyperprolactinemia is detected, treatment with dopaminergic agonists may be considered in women to increase live birth rates [2, 3].Since hyperprolactinemia is an easily treatable cause, most centers routinely test serum prolactin levels.
\nThere are few studies evaluating the association between vitamin D deficiency and RPL [2]. One of them showed increased prevalence of hypovitaminosis D in women with RPL, but it was unable to demonstrate cause-effect relationship [2, 54]. Thus, based on the significant prevalence of hypovitaminosis D in women with RPL and possible association with obstetric and fetal complications, the preconception counseling in these women may include prophylactic vitamin D supplementation [2].
\nChronic endometritis (CE) is defined as a persistent inflammation of the endometrial mucosa caused by the presence of bacterial pathogens in the uterine cavity [55]. Its prevalence in patients with RPL is approximately 12–13% [56]. The influence of CE on reproductive capacity is controversial, but many authors suggest that CE may negatively affect embryonic implantation [56]. Some studies suggest an infectious etiology with positive cultures in 75% of women with histologically confirmed CE, with the most common bacteria being Escherichia coli, Enterococcus faecalis, and Streptococcus agalactiae (77.5%) [57]. Most patients are asymptomatic, with pain on uterine or cervical mobilization being the most common clinical presentation [58, 59].
\nCE is histopathologically diagnosed as a lymphoplasmacytic infiltrate in the endometrial stroma [58, 59]. Immunohistochemistry for the marker present in CD138 plasma cells is used to improve diagnostic accuracy [60]. A diagnostic video hysteroscopy can help identify CE, with direct visualization of the endometrial cavity, which usually presents with mucosal edema, focal or diffuse endometrial hyperemia, or micropolyps. The sensitivity, specificity, and positive and negative predictive values of hysteroscopy in diagnosing CE were 86.36, 87.30, 70.37, and 94.82%, respectively [61].
\nUp to a few years ago, the uterine cavity was thought of as a sterile environment. Recently, there has been discussed that an imbalance of the uterine microbiota might compromise embryonic implantation or induce an abortion. Endometrial biopsy for next-generation sequencing (NGS) microbiota evaluation and etiological agent research can now be done through commercial kits [55]. However, further studies are needed to evaluate diagnostic efficacy and therapy on the reproductive outcomes.
\nSome studies suggest that treatment is related to increased live birth rates and reduced abortion rates [62]. There are several therapeutic options; the main ones mentioned in the literature refer to the use of doxycycline alone (100 mg, 12/12 hours orally, for 14 days) or the combination of metronidazole (250 mg, 12/12 hours orally, for 14 days) and ciprofloxacin (250 mg orally 12/12 hours for 14 days) [59].
\nThere is a growing acceptance of male etiological factors for RPL. Its screening consists of detailed sperm analysis. Excessive sperm DNA fragmentation is an important constraint to conception. Two meta-analyses have shown the association of gestational losses with high rates of sperm DNA fragmentation [63, 64]. The available tests for sperm DNA fragmentation index are the sperm chromatin structure assay (SCSA), the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), the Sperm Chromatin Dispersion test, and the comet assay.
\nSome clinical conditions are related to increased fragmentation of sperm DNA. High seminal plasma leukocyte concentration, systemic infections, varicocele, and smoking, among others, were related to spermatic DNA damage [65]. A Cochrane meta-analysis suggests that the use of antioxidants, including vitamins C and E, may have benefits for subfertile men with no apparent cause, improving sperm DNA fragmentation [66]. The generally recommended dose is 1 gram of vitamin C and 1000 IU of vitamin E per day for at least 2 months [67]. However, this effect is not yet established in patients with RPL. ESHRE determines that sperm DNA fragmentation research should be considered for explanatory purposes for RPL [2].
\nFor intracytoplasmic sperm injection (ICSI)-indicated couples, laboratory techniques may be performed to select sperm with lower DNA fragmentation rate, such as physiological intracytoplasmic sperm injection (PICSI) and intracytoplasmic morphologically selected injection (IMSI). However, the use of testicular sperm seems to improve fertilization, pregnancy, and live birth rates when compared to PICS and IMSI techniques [68]. Nevertheless, further studies are needed to identify the best method for selecting sperm to reduce abortion rates.
\nThe morphological analysis of sperm is another point to consider in cases of RPL. The presence of spermatozoa with structural anomalies may be associated with aneuploidy, resulting in aneuploid embryos that usually do not implant or are aborted. This is especially true in cases of globozoospermia and macrospermia, forms of monomorphic teratospermia—when all sperms have the same anomaly [69]. Infertility is generally associated with these cases, and the prognosis of IVF is reserved. Thus, when associated with abortion, IVF followed by embryonic biopsy for preimplantation genetic testing for aneuploidies (PGT-A) may be an option.
\nRecurrent spontaneous abortion is an entity with a multifactorial etiology, and in approximately 50% of cases, we did not identify the cause of the loss. This explains the large number of controversies regarding the investigation and treatment of the pathologies that lead to repeated losses.
\nDespite so much controversy, there are some points on which experts agree. Psychological support for couples is essential and is associated with a better prognosis in subsequent pregnancy. Undergoing through periodic consultations and ultrasounds especially during the period of previous losses reduces the stress of these couples. The woman’s age and number of previous losses are the most important factors in predicting the couple’s chance of having a live baby in the next pregnancy.
\nThere is a need for consensus among human reproduction societies on the tests that must be ordered and diagnostic criteria for all specialists to evaluate couples evenly. In this way, we will be able to evaluate the effectiveness of each available treatment, avoiding further financial burns, emotional disorders, and iatrogenesis for these couples.
\nThe authors have no conflicts of interest that are relevant to this report.
Supporting women in scientific research and encouraging more women to pursue careers in STEM fields has been an issue on the global agenda for many years. But there is still much to be done. And IntechOpen wants to help.
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