In vitro maturation of oocytes from stimulated IVF-ICSI cycles using autologous cumulus cell co-culture: A preliminary study.

PURPOSE OF THE STUDY: In stimulated IVF-ICSI cycles, follicles at different stages of maturation can be aspirated during oocyte pickup. Nowadays, only mature oocytes (metaphase 2 stage) are used and immature oocytes (germinal vesicle and metaphase 1 stages), which are judged unfit for fertilization, are non-used at day 0. In our IVF center, the rate of immature oocytes recovered is around 25%. A significant number of this precious resource is therefore non-used every day in IVF laboratories. The objective of our study was to evaluate the competence of our in vitro maturation autologous coculture method on the maturation and developmental potential of immature oocytes obtained from stimulated IVF-ICSI cycles, in order to obtain additional embryos for the couple as a rescue system to increase the changes of cumulative pregnancy. PATIENTS AND METHODS: This is a prospective study, carried out in the Reproductive Medicine and Biology Unit of the Amiens-Picardy University Hospital (France). It was included 14 couples, managed in IVF-ICSI in our center, from January to March 2020. Thirty-eight oocytes, identified as immature after cumulus-oocyte complexes (COC) stripping for ICSI, were placed in our in vitro maturation medium with the addition of autologous cumulus cells. Oocytes that had reached the metaphase II stage after a maximum of 36 hours of maturation were microinjected. The fertilization and embryonic development potential of the in vitro matured oocytes were compared to those of 148 in vivo matured "siblings" oocytes from the same oocyte retrieval, and then also compared to those of 127 in vivo matured oocytes from different patients (control group). MAIN OUTCOME MEASURE(S): Maturation rate, fertilization rate, early cleavage rate and developmental activity to blastulation rate. SECOND OUTCOME MEASURE(S): Embryo quality at cleavage and blastocyst stages, blastulation rate, and useful blastulation rate. RESULTS: No significant difference was found in the main and secondary criteria of the study compared to the "siblings" in vivo matured oocytes from the same oocyte retrieval. However, a significant difference was obtained on the rate of early cleavage and useful blastulation when our cohort was compared to mature in vivo oocytes from different patients (control group). CONCLUSION: This study has shown that after incubation in our in vitro maturation autologous cumulus cell co-culture with cumulus-oocyte cells, immature oocytes recovered during stimulated cycles can give rise to competent oocytes, i.e., capable of being fertilized, of cleaving, and of developing into embryos up to the blastocyst stage. Our study therefore seems to be in the direction of a favorable use of these immature oocytes obtained after stimulated IVF-ICSI cycles. The continuation of this study by including a larger number of oocytes is necessary in order to evaluate the real contribution of this technique in routine.

Efficacy of assisted reproductive technology after ovarian tissue transplantation in a cohort of 11 patients with or without associated infertility factors.

PURPOSE: IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor reproductive outcomes. The aim of this study was to evaluate the efficacy of ovarian tissue transplantation (OTT) followed by assisted reproductive technology (ART) in women with or without associated infertility factors. METHODS: This is a prospective cohort study with retrospective data collection including eleven women, four of whom having an infertility factor (IF), who had undergone OTT in one university center between 2005 and 2017, followed by ART in six in vitro fertilization (IVF) centers. RESULTS: In total, 25 of the 85 cycles initiated (29%) were canceled, resulting in 60 oocyte retrievals. Ninety-five oocytes were retrieved: 36 were abnormal or immature, 29/39 fertilized (74%) after ICSI and 13/20 (65%) after IVF. Thirty-five embryos were transferred in seven patients (5/7 patients without IF and 2/4 patients with IF). After ART, one patient with IF experienced two pregnancies, one resulting in a live birth. For all patients, pregnancy rates and live birth rates were 7.4% and 3.7% per embryo transfer, respectively. Nine pregnancies and four live births occurred after spontaneous conception in five patients without IF, none in the infertility group. CONCLUSION: This study confirms that IVF treatment in women with grafted frozen-thawed ovarian tissue is associated with poor outcomes. However, the chances of natural conception are high in women without IF. Patients with IF, without the possibility of spontaneous pregnancy, should be informed of poor reproductive outcomes after OTT followed by ART. TRIAL REGISTRATION NUMBER: NCT02184806.

Successful in vitro maturation of oocytes in a woman with gonadotropin-resistant ovary syndrome associated with a novel combination of FSH receptor gene variants: a case report.

Infertility due to Gonadotropin-Resistant Ovary Syndrome (GROS) is a rare type of hypergonadotropic hypogonadism. Here, we report an original case of GROS, associated with compound heterozygous follicle-stimulating hormone receptor (FSHR) variants, in a woman who achieved a live birth by in vitro maturation (IVM) of her oocytes. This 31-year-old woman consulted our assisted reproduction center for a second opinion after having been advised, because of pervasive high serum follicle-stimulating hormone (FSH) levels, to pursue in vitro fertilization (IVF) with donor oocytes. She presented with primary infertility and progressively prolonged menstrual cycles. Her serum FSH levels were indeed found to be high, but in discordance with a normal anti-Müllerian hormone (AMH) level and antral follicle count. Genetic investigation found the patient to be compound heterozygous for two FSHR variants: I160T, a known pathologic variant, and N558H, which has never been previously reported. As there was no ovarian response to high daily doses of exogenous gonadotropins, IVM was proposed to the patient with success and she finally delivered at term a healthy boy. Effects of the receptor variants were analyzed in heterologous cells. Whereas the I160T mutation blocked FSHR membrane trafficking and FSH-stimulated cAMP-dependent signaling in transfected CHO cells, the novel variant, N558H, functioned equivalently to wild-type FSHR in the assays employed. In conclusion, IVM should always be offered as a first-line therapy to infertile women presenting with GROS. The N558H variant discovered in FSHR is novel, but its functional significance, if any, is unresolved and merits further investigation as it may be associated with a recessive FSHR-related disorder.

[Genetic aspects of male infertility: From bench to clinic]. / Aspect génétique de l'infertilité masculine : de la recherche à la clinique.

OBJECTIVES: The objective of our manuscript is to review the current state of research on the genetics of male infertility, highlighting the genetic abnormalities that can lead to non-syndromic male infertility and genetic testing proposed to patients. It is intended primarily for clinicians and biologists of reproductive medicine. METHODS: A comprehensive review of the scientific literature available on PubMed was conducted using keywords related to male infertility and genetics. Since the first genes related to non-syndromic male infertility were identified after the 2000s, bibliographic research was conducted after this date. RESULTS: Thirty-three genes have been identified as responsible for non-syndromic male infertility. The evolution of techniques based on whole genome analysis has allowed the development of more successful methods in the identification of new genes and mutations inducing an infertility phenotype. Through this article, we propose, by concrete examples, a clinical approach for genetic tests considering the semen analysis alterations. CONCLUSIONS: The identification and characterization of these genes and the mutations responsible for certain infertility phenotypes allow better management and better treatment for patients as well as a better understanding of the physiopathological mechanisms of human gametogenesis.

Impact of alcohol and cigarette smoking consumption in male fertility potential: Looks at lipid peroxidation, enzymatic antioxidant activities and sperm DNA damage.

Alcohol intake and cigarette smoking are the major lifestyle factors with negative impact on fertility. We were interested to evaluate the negative impact of these factors on oxidative stress (OS), enzymatic antioxidant activity (EAO) of spermatozoa and on its DNA damage. This study included 108 male infertile patients with normal range of sperm conventional parameters but with unexplained infertility in assisted reproductive technologies programme. Firstly, OS was analysed based on lipid peroxidation (MDA) and EAO which included catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR). Secondly, we evaluated DNA fragmentation by TUNEL assay and chromatin decondensation by aniline blue colouration. The whole lot was divided into four groups: control (nonalcoholic and nonsmoker patients), alcohol group, smoking group and alcohol-smoking group. The results showed, in three last groups compared to control an increased CAT, SOD and GR activities with high MDA level especially in smoking and alcohol-smoking group. The latter showed the highest values of DNA fragmentation and chromatin decondensation (31% and 39%) to exceed DNA damage normal range. Indeed, smoking and alcohol intake lead to increase EAO due to long-term unbalanced antioxidant/oxidation ratio with high OS which cause consequently sperm DNA damage calling in need by urgency to change the lifestyle behaviour.

Detection of Loa loa Microfilariae in Follicular Fluid During Assisted Reproductive Technology: Case Report and Review of the Literature.

We report on a case of loiasis revealed during an assisted reproductive technology (ART) cycle. Loa loa could limit implantation outcome. We propose to focus on an ART strategy with frozen embryos to treat the patient before any transfer.

Effect of semen preparation technique and its incubation on sperm quality in the Moroccan population.

In in vitro fertilisation (IVF), sperm preparation as critical part and influencing the sperm quality is especially dependent on the chosen technique itself and incubation parameters including temperature and CO2. In this study, we compared firstly density-gradient centrifugation technique (DGC) to the adapted DGC using the sperm pellet of 80% fraction (DGC/80P) in order to improve the sperm yield. Secondly, this study led to evaluate different sperm incubation conditions based on temperature effect (room temperature (RT = 23°C) versus 35°C) and in the other hand, with or without 5% CO2 during 24 hrs. Based on evaluating sperm conventional parameters and the DNA damage using TUNEL assay, our result showed that DGC/80P increased sperm quality compared to DGC with 25% of improvement. For temperature incubation effect after 24 hrs, 35°C increased the DNA damage and decreased the sperm quality while RT could improve sperm motility by 38%. Moreover, the sperm incubation with 5% CO2 after 24 hrs realised a negative impact on sperm parameters and its DNA damage. Indeed, for current IVF practice, a good sperm quality can be maintained for several hours at room temperature, while the sperm preparation is processed using the DGC/80P without CO2.

Sperm global DNA methylation level: association with semen parameters and genome integrity.

Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity.

[Are patients in the postpartum period potential egg donors?]. / Les accouchées sont-elles de potentielles donneuses d'ovocytes ?

OBJECTIVES: In France, oocyte donation program is still underdeveloped because of lack of donors and this situation entails an important wave of cross border medical tourism to different European countries mainly Spain and Greece. In 2011, the General inspection of social affairs report recommended to the biomedicine agency to promote spontaneous oocyte donation via different channels of information to develop this national program. The main objective of this study is to assess the knowledge of women after baby delivery about oocyte donation. The second objective is the identification of ways to assure better information and to promote oocyte donation. PATIENTS AND METHODS: We conducted a prospective study with anonymous questionnaire distribution to women after delivery at obstetrics/gynecology department of the Regional University Hospital and Maternity-Children Unit "Victor-Pauchet" of Amiens, from December 2012 to January 2013. RESULTS: Two hundred and fifty-five questionnaires were distributed and 242 of them were analyzed (94.9%). About oocyte donation knowledge: 28% did not know it was possible, 45% did not know it was legal in France, 54% did not know who was concerned and 36% know that a treatment is necessary, 9% think that oocyte donation is paid and 10% it is non-anonymous. If 67% seems to be favorable to this initiative, only 35% could accept to realize it. About information efficiency, 88% think not to receive enough information, 64% would like to have more information. The health care professional wanted to give this information is an obstetrician (51%), a midwife (37%) and a nurse (12%). DISCUSSION AND CONCLUSION: Oocyte donation program is misoriented due to a lack of information. Obstetricians and midwives have an important educational and informative role to support oocyte donation. Specific strategy of communication and valuable targeted information are needed to motivate potential donor and achieve the objectives of the program.

Autologous embryo-cumulus cells co-culture and blastocyst transfer in repeated implantation failures: a collaborative prospective randomized study.

In repeated implantation failure, the co-culture of human embryos with somatic cells has been reported to promote the improvement of embryos quality, implantation and pregnancy rate. It was reported that feeder cells can be more beneficial to the oocyte and embryo by detoxifying the culture medium and supporting embryo development via different pathways. In this study, 432 patients, each with a minimum of three repeated implantation failures, were accepted for a prospective randomized study with or without autologous cumulus cell embryo co-culture and transfer at day 3 or day 5-6. We also investigated the expression of leukaemia inhibitor factor (LIF) and platelet activating factor receptor (PAF-R) on day 3 confluent cumulus cells. The statistic analysis of the data showed significant difference of implantation and clinical pregnancy rates between classical culture and day 3 compared with co-culture and day 5-6 transfer. The molecular analysis showed that cumulus cells express the LIF and the PAF-R genes and confirmed the possible positive role of growth factors and cytokines in early embryo development. Embryo co-culture systems with autologous cells can be beneficial in routine in vitro fertilization for embryo selection and implantation improvement. More molecular investigations need to be done to improve elucidation of the complex dialogue between the embryo and feeder cells prior to implantation and to understand the involved biological function and molecular process during embryo development.

Natural cycle IVF and oocyte in-vitro maturation in polycystic ovary syndrome: a collaborative prospective study.

In-vitro maturation (IVM) was performed in 350 cycles for 262 unstimulated patients diagnosed with polycystic ovary syndrome who were primed with human chorionic gonadotrophin (HCG) before oocyte retrieval. In order to improve nuclear and cytoplasmic maturation, growth hormone was added to the maturation medium. Oocytes were recovered in 94.8% of the cycles, with a mean number of nine cumulus-oocyte complexes retrieved. Within 28 h, 62% of the oocytes reached the metaphase II (MII) stage, and 17.6% were MII after a further 20 h in culture. An ongoing pregnancy rate of 15.2% was obtained, but with a high miscarriage rate, 28% of the total with a positive betaHCG test assessed after embryo transfer. Cytogenetic and DNA fragmentation analysis of the embryos was not fundamentally different from what is classically observed in routine IVF. This observation implies that the results are not necessarily due to compromised oocyte quality after IVM, and that endometrial receptivity should also be considered, especially in IVM cycles where the follicular phase is dramatically shortened.

De novo subtelomeric deletion additional to an inherited apparently balanced reciprocal translocation.

OBJECTIVE: We describe the analysis of an apparently balanced inherited reciprocal translocation in a fetus presenting with multiple congenital abnormalities, characterize the structural chromosome rearrangement, and report an unexpected additional imbalance to the inherited rearrangement. METHODS: DNA microarray was used to screen for genomic imbalance in subtelomeric and interstitial critical regions. High-resolution comparative genomic hybridization was used to screen for genomic imbalance at a genome-wide level. Fluorescence in situ hybridization using whole-chromosome painting and specific probes was used to characterize the inherited translocation, and the size of the de novoadditional deletion. RESULTS: An unexpected additional deletion was found in 7qter on derivative 10 of the inherited maternal reciprocal translocation t(7;10)(q11.23; p14). CONCLUSIONS: We show the usefulness of genome-wide and specific molecular cytogenetic techniques to explore apparently balanced rearrangements.

In-vitro maturation of oocytes: biological aspects.

Natural cycle and in-vitro maturation (IVM) of oocytes are becoming interesting alternatives to classical assisted reproduction technology approaches for patients, especially in those at high risk for ovarian hyperstimulation syndrome or with poor ovarian reserve. More than for their clinical and biological indications, natural cycle and IVM of oocytes can also be considered as good social and economic alternatives to the classical IVF treatment, based on their financial cost-effectiveness with exclusion of expensive medications. To be successful, IVM must entail both nuclear and cytoplasmic maturation, and its maturation and success rates are affected by the number of collected cumulus layers, the degree of atresia and the maturation rate between 24 and 48 h. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. This complex process requires a better definition of each contributing factor affecting oocyte development and the resulting embryo quality. The clinical aspects of IVM have been documented earlier; the present paper will mainly focus on the biological aspect of oocyte maturation in vitro and the quality of derived embryos.

Array comparative genomic hybridization profiling of first-trimester spontaneous abortions that fail to grow in vitro.

OBJECTIVES: Cytogenetic analysis of spontaneous abortion samples can be limited by culture failure. Failure to grow in vitro has traditionally been suspected to be due to in vivo death of tissue associated with spontaneous abortion (SAB) or simply technical factors of growth in culture. METHOD: We used array comparative genomic hybridization (array CGH) to investigate chromosomal imbalances in products of conception that failed to grow in vitro. RESULTS: Our data on 26 cases of SABs that failed to grow in culture are compared and contrasted with published data on cytogenetic findings following in vitro culture. The results revealed abnormalities uncommonly seen by classic cytogenetic methods. These abnormalities include high rates of double aneuploidy and autosomal monosomy. The data taken together suggest that classic cytogenetics of spontaneous abortion may yield normal karyotypes or selected abnormal karyotypes that permit cell proliferation in vitro while Array CGH detects other abnormalities. CONCLUSION: Array CGH is becoming an important clinical assay for unbalanced chromosome abnormalities whether cells grow in culture or not and in cases of analysis on one or few cells.

De novo interstitial direct duplication of Xq21.1q25 associated with skewed X-inactivation pattern.

Genotype-phenotype correlation in women with an abnormal phenotype associated with a duplication of the long arm of the X chromosome remains unclear. We report on prenatal diagnosis and follow-up of a girl with an Xq duplication and dysmorphic features. The abnormal phenotype included growth retardation, hypotonia, and nystagmus. In order to improve the resolution of the cytogenetic analysis, we used both conventional and array-based comparative genomic hybridization to perform a global molecular cytogenetic analysis of the genome. These molecular cytogenetic analyses showed a direct duplication Xq21.1 --> q25 without other chromosomal abnormalities. This duplication was originating from the paternal X chromosome. Moreover, a skewed X-inactivation pattern was observed leading to a partial functional disomy of the chromosomal region Xq21.1q25. This report and review of the literature suggest that functional disomy for chromosome X could explain the abnormal phenotype. In prenatal diagnosis, this can have implication for patient management and genetic counseling.

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques.

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium-magnesium-free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group.

Assessment of DNA fragmentation and aneuploidy on poor quality human embryos.

In human assisted reproduction, low embryo quality due to retarded growth and abnormal cellular morphology results in fewer embryos suitable for transfer. This study aimed to assess the extent of DNA fragmentation and aneuploidy in spare slow growing or arrested human embryos. In 19 assisted reproduction cycles, a total of 57 embryos unsuitable for embryo transfer were used for simultaneous apoptosis and aneuploidy assessment. Among them, 31 (54.3%) showed DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) analysis. Among 26 embryos that were negative for TUNEL, interpretable fluorescence in-situ hybridization (FISH) results were obtained for 25 embryos (96.2%). Sixteen embryos were detected to be chromosomally abnormal (64.0%); three were found to be chaotic, six had complex aneuploidy, six had complete monosomy and one was polyploid. The results show that a high level of DNA fragmentation and aneuploidy are common in embryos with slow growth and/or low quality. More detailed studies are needed to assess the effect of factors such as ovarian stimulation regimens and in-vitro culture conditions. Moreover, application of simultaneous TUNEL and FISH techniques can be informative regarding DNA integrity and aneuploidy.

[A preliminary study to assess the value of the DNA chips SpectralChip to detect subtle constitutional chromosome imbalances]. / Evaluation de la puce à ADN SpectralChip pour la détection des déséquilibres chromosomiques observés en pathologie constitutionnelle.

Comparative genomic hybridization on a microarray (microarray-CGH) allows to detect genomic chromosome imbalances. In order to assess its value to detect small chromosome imbalances observed in a clinical setting, using a DNA chip available commercially (Spectral Genomics, Houston, Texas, USA), we studied the DNA of 9 patients carrying a well characterized chromosome imbalance and the DNA of 11 patients where cytogenetic techniques such as high resolution banding karyotype, FISH using subtelomeric probes and comparative genomic hybridization on metaphase chromosomes conclude to a normal and/or balanced karyotype. A result was obtained for 19/20 patients. Failure of hybridization was observed for one patient. For all the other cases the sex of patients was correctly identified. Microarray-CGH was able to correctly diagnose the chromosome imbalance in 6/8 patients carrying such a defect i.e 9/11 imbalances (deletion or duplication) were detected. No chromosome imbalance was observed in 11 patients considered normal and/or balanced using cytogenetic techniques. Several clones were found to be polymorphic and required FISH studies to eliminate duplication or deletion. In conclusion, we think that this commercially available DNA chip might be useful to screen for chromosome imbalances. However, technical improvements are still necessary before using it in a clinical setting. Also, further studies are necessary to assess its sensitivity and specificity.

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes.

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19-25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART.