text:
The effect of follicle size and homogeneity of follicular development on the morphokinetics of human embryos

Purpose Our aim was to investigate follicular size (large, ≥17 mm and small, <17 mm) at the time of OPU and homogeneity of follicular development (homogenous development: follicles being present in a homogenous spread of all sizes; heterogeneous: a predominance of small and large follicles) by analysing the morphokinetics of embryo development. Methods In this prospective cohort study, 2526 COCs belonging to 187 patients were cultured to day 5. Embryos were evaluated morphokinetically. Four subgroups were defined: large follicles from heterogeneous cycles (LHet) and homogenous cycles (LHom) and small follicles from heterogeneous cycles (SHet) and homogenous cycles (SHom). Results Rates of fertilization, blastocyst formation and top and good quality blastocysts were found to be significantly higher in embryos from the LHom group (p < 0.001; p < 0.001; p < 0.001). Small follicles from both homogenous and heterogeneous cycles had significantly lower blastocyst formation and top and good quality blastocyst rates (p < 0.001; p < 0.001). Embryos from SHet had significantly more direct cleavages (p = 0.011). Time to reach blastocyst was shorter in SHom than LHet and LHom (p = 0.002; p = 0.027, respectively). However, once the blastocyst stage was achieved, implantation rates were not significantly different between subgroups, the highest rate being observed in the LHom group. Multivariable analysis revealed that homogeneity of follicular development and follicular size had a significant effect on blastocyst development and quality (p = 0.049; p < 0.001, respectively). Conclusion Follicular dynamics, illustrated by follicular size and homogeneity of follicular development, influence early human embryo development. Patterns of follicular growth have an impact on embryo quality and viability which is reflected in morphokinetic variables. Electronic supplementary material The online version of this article (doi:10.1007/s10815-017-0935-1) contains supplementary material, which is available to authorized users.

Introduction
This study was undertaken as a result of clinical practitioner observations in an assisted reproductive technology (ART) setting which led to questions regarding a possible relationship between follicular size, homogeneity of follicular development and reaching blastocyst stage and clinical outcomes. To the best of our knowledge, these questions had not so far been studied.
Our study aimed to investigate the clinical relevance of two common controlled ovarian stimulation (COS) parameters: follicular size (large and small) at the time of oocyte pickup (OPU) and homogeneity of follicular development (homogenous development: follicles being present in a homogenous spread of all sizes; heterogeneous: a predominance of small and large follicles) by analysing the morphokinetics of embryo development and to evaluate clinical outcomes.
The reason why some oocytes have better developmental capacity than the other oocytes developed in the same Electronic supplementary material The online version of this article (doi:10.1007/s10815-017-0935-1) contains supplementary material, which is available to authorized users. cohort is not known yet. The mechanism underlying the individual response of antral follicles to exogenous gonadotropin has not yet been clearly determined. However, it is known that early antral follicles do not necessarily grow coordinately in response to exogenous gonadotropins to reach simultaneous functional and morphologic maturation, and not necessarily all FSH responding follicles yet have enough LH receptors to respond to the maturation signal introduced by hCG [1,2]. Moreover, in the GnRH antagonist cycles, a physiological increase in the FSH level during the luteal-follicular transition phase provokes a heterogeneous follicular development leading to a slightly lower maturation rate when compared to agonist cycles. During the early follicular phase, early antral follicles present noticeable size heterogeneities that may be amplified during COS [3]. Thus, multifollicular growth may result in heterogeneous size of follicles, variable growth rate and also may cause secondary and tertiary cohorts [4][5][6][7].
There are studies in the literature regarding embryo development and follicular size indicating that better oocytes are obtained from large follicles [8][9][10][11][12][13]. However, Nivet et al., when studying the impact of follicular size on oocyte quality as measured by embryonic development, demonstrated that medium size follicles yield a better percentage of transferable embryos [14].
Oocyte size is a key factor for meiotic competence while developmental competence is not present before the full size is reached. Intrinsic oocyte quality determines the oocyte's ability to overcome meiotic arrest and progress to the metaphase II (MII) stage (meiotic competence) and to undergo fertilization and support early embryonic stages (developmental competence) [15]. Nuclear and cytoplasmic maturation are characterized by an increased oocyte diameter, chromatin condensation, transcriptional quiescence and also mitochondrial aggregation towards the nucleus [16,17]. Therefore, MII oocytes may, because of poor cytoplasmic maturity, result in suboptimal rates of embryo development.
The development of incubators with built-in time-lapse technology has enabled continuous non-invasive monitoring of embryo development from fertilization to blastocyst stage and the possibility of appraising the precise timing of embryonic cell divisions [18][19][20][21][22]. Many studies have tried to connect, more or less conclusively, embryo development, timing of mitotic divisions, embryo viability and implantation. However, time points indicating precise embryo cleavages were shown to be affected by extrinsic factors such as ovarian stimulation protocols, culture conditions, fertilization method and also by patient-specific factors [23][24][25][26][27][28].
To the best of our knowledge, no previous studies have investigated both follicular size and homogeneity of follicular development by analysing the morphokinetics of embryo development.

Patients
This prospective cohort study was registered at clinicaltrials.gov (NCT02230449) and obtained an ethical approval from the institutional review board (23/06/2014-19). It was conducted in a private IVF clinic between July 2014 and September 2015. The analysis was based on a total of 2526 cumulus oocyte complexes (COCs) belonging to 187 patients with culture until day 5 since 13 patients were excluded (one premature ovulation, one oocyte maturation defect, two low fertilizations, three fertilization failures, two cleavage-stage developmental arrests, four OHSS/freeze-all). In the cases of low fertilization, one patient had only one fertilized oocyte which was abnormal (three pronuclei (PN)) and in the other case, despite more than eight COCs being retrieved, there was only one MII oocyte which presented only one PN and did not cleave. In the two cases of cleavage-stage arrests at the two-cell stage, the blastomeres were significantly different in size, possibly indicating abnormality. The patients included in this study presented various infertility causes and the mean female age was 31.1 years. All protocols were approved by the institutional review board and all patients gave their informed consent prior to their inclusion in the study. Patients were selected based on inclusion criteria (age ≤39 years, body mass index (BMI) <30 kg/m 2 , ≥8 COCs retrieved, <2 previous treatment cycles, hCG trigger) and exclusion criteria (recurrent pregnancy loss, severe endometriosis, PGD or PGS, COC > 24, embryo transfer (ET) < day 5, PCOS, uterine anomaly, severe sperm morphological abnormality such as dominantly macrocephalic or globozoospermic sample or cryptozoospermia, ≤1million motile sperm cells in total ejaculate). All embryos were obtained after fertilization by intracytoplasmic sperm injection (ICSI) and were part of our standard ART program. Embryo development was recorded using time-lapse technology (EmbryoScope™ time-lapse system, Vitrolife, Göteborg, Sweden).

Ovarian stimulation
The baseline estradiol, LH and progesterone levels were evaluated on cycle day 2, and baseline ultrasound scans were performed on the same day. Depending on the BMI, antimullerian hormone (AMH) level, basal antral follicle count and the history, if any, of a previous response to gonadotropins, recombinant FSH (rFSH; Gonal-F®; Merck Serono, Switzerland) was used for ovarian stimulation at a dosage of 150 to 225 IU. The standard dose was 150 IU, when necessary however, depending on body mass index, 225 IU was started. From the fourth or fifth day of rFSH therapy onwards, patients were monitored daily or every other day for hormone levels (estradiol, LH and progesterone whenever needed) and follicular measurements. A daily administration of 0.25 mg GnRH antagonist (Cetrotide®; Merck Serono, Switzerland) was administered when the size of the follicle was at least 12 mm, but never exceeding 13 mm. Follicular maturation was achieved by using 250 μg recombinant hCG (Ovitrelle®; Merck Serono, Switzerland) when at least three follicles reached a minimum mean diameter of at least 17 mm. Transvaginal ultrasound-guided oocyte retrieval was scheduled for 36 h later.

Follicular size
Each follicular aspiration was performed by the same doctor in order to reduce any possible inaccuracy of measurement to a minimum. Follicles <17 mm at the time of OPU were classified as small while those ≥17 mm were classified as large. Oocytes coming from large and small follicles were incubated separately.

Homogeneity of multifollicular growth
The data was also evaluated according to the homogeneity of follicular development. Follicles being present in a homogenous spread of all sizes from large (>20 mm) to intermediate (17-20 mm) to small (<17 mm) was considered to be homogenous development, whereas a predominance of large (>20 mm) and small (<17 mm) follicles was considered to be heterogeneous. Each case met the criteria for trigger of a minimum of three follicles of at least 17 mm. Four subgroups have been defined according to the follicular size and cycle homogeneity: small follicles/heterogeneous follicular development (SHet), small follicles/homogenous follicular development (SHom), large follicles/heterogeneous follicular development (LHet) and large follicles/homogenous follicular development (LHom).

Oocyte retrieval, denudation and ICSI
On the day of OPU, follicles were aspirated separately and COCs were washed in human tubal fluid medium (HTF; Life Global®, Brussels, Belgium). The gynaecologist who performed the pickup informed the embryology laboratory of the size of follicle (large ≥17 mm /small <17 mm) from which each oocyte was derived. One embryologist retrieved oocytes with a second embryologist assisting to ensure smooth operation and to document the process of isolation, identification and positioning of COCs in the culture dish.
The COCs were then incubated for 3.5 h at 6% CO 2 , 5% O 2 and 37°C before denudation, which was carried out by mechanical pipetting in ICSI Cumulase® (Origio, Måløv, Denmark). Each COC was denuded separately and the maturation status was then determined. Next, oocytes were allowed to incubate for an additional 30 min. ICSI was then performed in an HTF medium with HEPES (Life Global®, Brussels, Belgium) at ×400 magnification using Olympus IX70 and Olympus IX71 inverted microscopes.

Embryo culture and incubation
Each of the 12 individual wells of the EmbryoSlide® culture dish was filled with 25 μl of a single step culture medium (Life Global®, Brussels, Belgium), supplemented with 10% Plasmanate (Life Global®, Brussels, Belgium), and all wells were covered with an overlay of 1.5 mL paraffin oil (Life Global®, Brussels, Belgium). Following ICSI, injected oocytes were positioned in the wells of the slide, which was placed in a time-lapse incubator (EmbryoScope™) at 6% CO 2 , 5% O 2 and 37°C for 5 days until embryo transfer. The culture medium was refreshed on the afternoon of day 3 by replacing the incubated slide with a new pre-equilibrated slide prepared as described above. Image stacks were acquired at seven focal planes every 15 min, and data were continuously transferred to an external computer, EmbryoViewer® workstation (Vitrolife, Göteborg, Sweden). Embryo development was annotated by one investigator and cross-checked by two other assessors.

Time-lapse evaluation and embryo scoring
Morphokinetic variables for all cleavage events up to the expanded blastocyst stage were annotated. All relevant events (fertilization, cleavages, morula and blastocyst formation) were checked on a daily basis, and time of cleavage to two-cell embryo (t2) and subsequent divisions t3, t4, t5, t6, t7, t8 and t9+ were recorded in the EmbryoViewer® workstation. The time of all mitotic events was expressed as hours post-ICSI. In order to minimize the variation of ICSI time within oocytes of one patient, ICSI was split between two embryologists above ten oocytes. Therefore, the maximum ICSI duration did not exceed 15 min, which is below the default time interval of each picture taken by the camera of the EmbryoScope™ system. tM was annotated at the end of the compaction process, when compaction was observed to be full with no apparent cell contours. tSB marks the initiation or start of blastulation, the first frame when initiation of a cavity formation is observed. tB indicates a blastocyst, where the ICM and the cavity are formed. tEB shows an expanded blastocyst with 50% thinning of the zona pellucida. Blastocysts were scored according to Gardner's classification (114-120 h post-ICSI) and selected for transfer based on the final morphology and the score obtained from the morphokinetic ratios published by Çetinkaya and colleagues (CS2-8 = ((t3-t2) + (t5-t4)) / (t8-t2); CS4-8 = (t8-t5) / (t8-t4)) [29].

Embryo transfer
After embryo transfer, for luteal phase support, patients received a twice daily dose of progesterone gel administered intravaginally (Crinone® 8%; Merck Serono, Switzerland). When pregnancy occurred, a daily dose was continued until the 10th week of gestation. Fourteen days after pickup, serum β-hCG was measured. At 7 weeks, a transvaginal ultrasound was performed to monitor early pregnancy. The implantation rate was calculated by dividing the number of implanted embryos by the total number of transferred embryos.

Power calculation
An earlier pilot study revealed that top and good quality blastocyst rates for large and small follicles were 45 and 32%, respectively. A power analysis indicated that, for an alpha level of 0.05 and a beta level of 0.20 (power = 0.80), 438 fertilized oocytes were sufficient to detect a significant difference between follicle size groups.

Statistical analysis
Demographics of patients were reported as minimum, maximum, mean ± sd and 95% confidence interval of the mean. Due to the dependent nature of the data, generalized linear mixed models with logit link function were conducted to analyse the differences of binary variables between four groups, namely LHom, LHet, SHom and SHet. Generalized linear mixed models with linear link function were conducted to analyse the differences of continuous variables between the four subgroups mentioned above.
Generalized linear mixed models with logit link function were conducted to analyse the possible effects of factors on the rate of top and good quality blastocysts. First, five models were conducted separately to test the effects of age, AMH, BMI, homogeneity and follicle size on the rate of top and good quality blastocysts. Second, a multivariable model was conducted, where variables with p < 0.20 significance level at univariable analysis were introduced as independent variables. A p value of <0.05 was considered statistically significant. All statistical analyses were performed using the MedCalc Statistical Software version 13.2.0 (MedCalc Software bvba, Ostend, Belgium) and R version 3.3.2.

Results
Cycle outcomes in the study cohort A total of 187 patients were prospectively involved in the analysis, using strict inclusion criteria, which allowed the study of a homogeneous, young, infertile patient population with a low BMI and a good ovarian reserve (mean female age 31.1 ± 4.1, AMH 3.1 ± 2.0 ng/mL and BMI 23.6 ± 3.0 kg/m 2 ). An average of 14 COCs was collected, 10.1 of which were at MII stage (maturation rate 73.6%). The fertilization rate after ICSI was 76.6%. The fertilized oocytes (71.7%) became grade 1 or 2 embryos on day 3 and 37.2% top or good quality blastocysts on day 5 ( Table 1). The clinical and ongoing  Table 1).

Outcomes according to follicular size and homogeneity of follicular development
Maturation and fertilization rates of oocytes deriving from large follicles were significantly higher than those deriving from small follicles. When subcategorizing the data, large follicles developed in homogenous cycles (LHom) had better outcomes than large follicles developed in heterogeneous cycles (LHet), small follicles in homogenous cycles (SHom) and finally small follicles in heterogeneous cycles (SHet). Also, rates of grade 1 and 2 embryos on day 3, blastocyst formation, top and good quality blastocysts were gradually higher in the same order mentioned above.

Morphokinetic analysis of follicular size and homogeneity of follicular development
The kinetics of embryos derived from small and large follicles was evaluated according to cycle homogeneity. Statistically significant differences were found beginning from the first cleavage (t2) until the expanded blastocyst time (tEB) except for t3 and t4. Embryos from small follicles developed faster than embryos originating from large follicles, for all cleavage timings. Embryos from SHet developed faster, except for t9 and tB ( Table 2). tB time was significantly different between follicular size and homogeneity groups (p = 0.001). Generalized linear mixed models were conducted for all comparisons. Bonferroni corrected post-hoc results were reported. Time of cleavage to two-cell embryo (t2), and subsequent divisions t3, t4, t5, t6, t7, t8 and t9+, were annotated. tM, tB and tEB are times to achieve a morula, a blastocyst and an expanded blastocyst, respectively. CS2-8 = ((t3-t2) + (t5-t4)) / (t8-t2); CS4-8 = (t8-t5) / (t8-t4) [28] tSB time for start of blastulation *p < 0.05 Bonferroni corrected post-hoc analysis revealed that tB time was shorter in the SHom group than LHet or LHom groups (p = 0.002; p = 0.027, respectively), whereas embryos developing from the SHet group had a tB shorter than embryos coming from the LHet group (p = 0.002). When median values were compared, the time to achieve a blastocyst for a small follicle was 1.4 h faster than for a large follicle (p = 0.0036) (Fig. 1). Another difference observed, when embryos developing from small and large follicles in homogenous or heterogeneous cycles were compared, was the significantly different rate of direct cleavages (t3-t2 < 5 h) (p = 0.011) ( Table 2). Bonferroni corrected post-hoc analysis revealed that the direct cleavage rate was higher in the SHet group (26.7%) than in the LHet (18.2%) (p = 0.016) and in the LHom groups (16.8%) (p = 0.006).

Embryo developmental arrests according to follicular size
Embryos originating from small follicles had a higher arrest rate than embryos originating from large follicles when analysing those having a first cleavage t2 (Fig. 2).
Cumulative developmental arrest rates of embryos originating from small and large follicles were calculated by adding all arrested embryos up to that time point for each category divided by embryos having achieved the two-cell stage. When looking at the time to achieve the eight-cell stage (t8), 91.4% of embryos developing from large follicles reached t8, compared to only 86.9% of embryos developing from small follicles with a 4.5% lower arrest rate in the former group (p = 0.0032) (Fig. 2). No significant differences were found between the developmental arrest rates of embryos from t8 to expanded blastocyst (tEB).

Implantation rates according to follicular size and homogeneity of follicular development
Although not statistically significant, embryos developing from the LHom group had a higher implantation rate when compared first to SHom, second to LHet and finally to SHet groups (58.1, 47.5, 36.7, 35.2%, respectively; p = 0.237) ( Table 2).

Univariable and multivariable analyses of factors affecting blastocyst quality
Univariable analyses revealed that AMH, homogeneity of follicular development and follicular size had a significant effect on blastocyst quality (p = 0.010; p = 0.018; p < 0.001,  (Table 3).
Multivariable analysis revealed that AMH, homogeneity of follicular development and follicular size had a significant effect on blastocyst quality (p = 0.010; p = 0.018; p < 0.001, respectively). One unit increase in AMH level resulted in a 1.

Discussion
To the best of our knowledge, this study describes for the first time the impact of follicular dynamics, illustrated by follicular size and homogeneity of follicular development, on early  Generalized linear mixed models were conducted for univariable and multivariable analyses *p < 0.05 human embryo development. One of the main findings emerging from this study is that a significantly higher number of good and top quality blastocysts suitable for transfer and freezing were observed in the LHom group. Although not significantly different between the subgroups, the highest implantation rate was also observed in the LHom group. Embryos originating from the LHom group outperformed significantly those from the SHom group in terms of maturation, fertilization, blastocyst formation and top and good quality blastocyst rates. Also, embryos originating from the SHet group showed significantly higher rates of developmental arrest or abnormal morphokinetic patterns such as direct cleavages, when compared to large follicles. However, once an embryo originating from a small follicle achieved the eightcell stage, no difference in the developmental progression up to the blastocyst stage was observed when compared to embryos originating from large follicles. In order to control for potential confounders, a multivariate analysis was conducted, revealing that in addition to AMH level, homogeneity of follicular development and follicular size had a significant effect on embryo developmental competency, namely the top and good quality blastocyst rate.
In our study, selection of blastocysts for transfer was done according to the score obtained from the morphokinetic ratios published by Çetinkaya and colleagues and on the final morphology [29]. The ratios used in this algorithm do not favour the preferential selection of fast developing embryos, but require a synchrony in mitotic divisions. This is in line with the recent discussion that the speed of development needs to be within an optimal range, meaning that both too slow and too fast development will impact success rates [30]. The Bquiet embryo hypothesis^postulates that early embryo viability is associated with a relatively stable-low metabolism [31]. The data on which this hypothesis was based were drawn from measurements on the depletion and appearance of amino acids from the culture medium. However, extending evidence on metabolic activity to the kinetics of pre-implantation embryo development, as recently reviewed by Leese and colleagues, may lead us to postulate that both too slow and too fast development result in lower success rates, due to non-optimal metabolic and/or genetic phenotype [30]. Therefore, intermediate kinetic ranges may be more physiological and may give better outcomes. Hence, as mentioned by these authors, a nonoptimal metabolic status reflects the physiological and bioenergetic status of the embryo as a function of mitochondrial health. Because the mitochondrial pool, RNAs and proteins of an embryo were derived from the oocyte, the quality of follicular growth directly impacts embryo quality and viability. This is reflected in morphokinetic variables of early embryo development. This could also explain why embryos derived from small follicles achieved faster the blastocyst stage than those from large follicles, implying that in practice, the selection of embryos based on their rapidity may erroneously deselect embryos developing from large follicles which may have a higher implantation potential.
One other possible explanation for the different early developmental characteristics of embryos derived from small or large follicles could be a difference in cytoplasmic competence. Small follicles containing MII oocytes may have achieved nuclear competence but not necessarily cytoplasmic competence. Also, cytoplasmic maturation has recently been shown to be associated with mitochondrial distribution in mouse and human oocytes [15,32].
The quiet embryo hypothesis also postulates that a low oxidative phosphorylation rate from the zygote to the morula stage limits reactive oxygen species production thus maximizing viability [33][34][35]. Oxidative stress can arise by the oxidative phosphorylation carried out by mitochondria and by the endoplasmic reticulum stress signalling [35]. We can speculate that the high bioenergetic level of small follicles leads to a precocious burnout reflected in morphokinetics, blastocyst and top and good quality blastocyst and implantation rates, thus inducing higher rates of developmental arrest and direct cleavages.
In conclusion, the highest rate of top and good quality blastocysts was achieved in embryos obtained from large follicles in homogenous cycles. A higher rate of direct cleavage and developmental arrest were observed in embryos obtained from small follicles. These findings can provide helpful information to both clinician and patient regarding the number of usable blastocysts likely to result in any particular cycle.
Compliance with ethical standards This prospective cohort study was registered at clinicaltrials.gov (NCT02230449) and obtained an ethical approval from the institutional review board (23/06/2014-19). All patients gave their informed consent prior to their inclusion in the study.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

Background Human adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells. Methods After approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins. Results In particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins. Conclusions We concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.

the interactions, biology and functions of these cells are more natural like [9]. Amongst various scaffolds, fibrin scaffold is a natural biopolymer which could be prepared from plasma. Moreover, it is a biocompatible and easily polymerized polymer capable of fusing cells and active biological molecules. Fibrin could provide the conditions for direct the cell phenotype and differentiation and could act as scaffold for tissue engineering [10]. 5-azacytidine (5-Aza) was one of the first inducers widely used to differentiate BMSCs into cardiomyocytes [11]. However, despite successful rabbit or mouse ADSCs differentiation, 5-Aza failed to trigger human ADSCs differentiation into contractile cardiomyocyte-like cells [12][13][14]. These deficiencies harbored by 5-Aza have lead researchers to use alternative agents like histone deacetylase inhibitor (HDAC inhibitors) such as trichostatin A. TSA is an organic compound that at first was used as an antifungal but later, its anticancer properties were discovered. It specifically inhibits the histone deacetylase families (HDAC) [15,16]. Human ADSCs treated by TSA indicate more expression of cardiac genes [16].
Although the ADSCs have been shown to have the potential for differentiation into cardiomyocytes and vascular cells, a highly efficient differentiation protocol is yet to be reported [17,18]. Therefore, in this study, we investigated this issue and found that TSA with scaffold is able to increase differentiation of hADSCs into cardiocyte.

Cell isolation
All materials were prepared from Sigma Company (Sigma-Aldrich, St. Louis, MO, USA) unless stated otherwise. Ethical approval was obtained from the Institutional ethical review board (approval number; IR. KMU. REC. 1394. 639) at Kerman University of Medical Sciences, Kerman, Iran. Human adipose-derived stem cells (hADSCs) were isolated from three 45 ± 3 old years women after a written consent had been obtained, using a standard cell culture protocol as previously described (article in review). Briefly, for primary culture of hADSCs, the adipose tissues were sliced into approximately 4 mm pieces and after washing with phosphate buffer saline (PBS) containing antibiotics, they were incubated with 0.5 mg/ml collagenase type IV, then centrifuged and the supernatant was removed and the cell pellet was suspended in culture medium containing; DMEM/F12 supplemented with 5% (v/v) FBS penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin-B (2.5 µg/ml) and cultured on plate at 37 °C and 5% CO 2 . After 24 h, the medium was changed with fresh medium. The medium was replaced every 3 days until the cells reached an approximately 80% confluence.

Fabrication of fibrin scaffold and cell encapsulation
For preparation of fibrinogen solution, fresh frozen plasma (FFP) was obtained from Iranian Blood Transfusion Organization. FFP was thawed in water bath at 37 °C and then 10 ml of it was mixed with 10 ml of Protamine sulfate. The obtained solution was centrifuged at 1800g for 10 min. The supernatant was collected and 0.1 M Sodium citrate was added to the tube. Then, the fibrinogen solution was filtered by a 0.2 µm filter and prepared for usage as cell culture. Mean final fibrinogen concentration was 60 mg/ml. For encapsulation of the hADSCs into the fibrin gel, 200 µL of fibrinogen solution was added to a 5 cc syringe containing 1 × 10 5 cells in 50 µl CaCl 2 (50 mM) and 50 µl thrombin solution (Sigma-T6884, Thrombin from human plasma) (40 IU/ml), and allowed to polymerize for 10 min at 37 °C, CO 2 incubator. Thus, hADSCs can be embedded in 3D fibrin gels. When the polymerization has ended and solution became gel, it was carefully immersed in a single well of a 6-well plates containing 2000 µl DMEM/F12 supplemented with 10% FBS, 1% antibiotic solution and 200 µl tranexamic acid, as an anti-fibrinolytic agent, then incubated at 37 °C in a humidified atmosphere containing 5% CO 2 for different time points of growth after initial seeding.

Scanning electron microscopy (SEM)
The scaffold was washed with PBS and fixed with 2.5% glutaraldehyde at room temperature. After rinsing the scaffold with PBS and then with double deionized water (DDI), the fibrin scaffold was immersed in liquid nitrogen and kept in a freezer dryer (Super Modulyo, Edwards) [19]. The samples were coated with gold for 180 s by a sputter coater (SC7620, Emitech, UK) and observed under Scanning Electron Microscope (SEM, AIS2100, Seron technology, South Korea) at an accelerating voltage of 20 kV. The diameter of the fibers was calculated from SEM images by image analysis software (Image J, NIH, USA).

Cell cycle analysis
Cell cycle analysis was performed by PI staining. In the first step, the cells from the fibrin scaffold and plate cell culture were detached at the 7th day after initial seeding. Then 1 × 10 6 cells were put in a 1.7 ml tube and a solution containing 0.25 g sodium citrate, 0.005 g ribonuclease A, 0.025 g PI, and 0.75 ml Triton X-100 was added in 250 ml distilled water and incubated at 4 °C for 30 min. The cells were evaluated by FACS, and percentage of the cells in each phase of the cell cycle (G0/G1, S, and G2/M phases) was calculated using the FlowJo software, version 7.5 (Tree Star, Inc., San Carlos, CA).

Induction of cardiac differentiation
In 2D group, 1 × 10 4 cells/cm 2 were seeded in cardiogenic medium, containing DMEM/ F12 supplemented with 100 IU/ml penicillin, 50 µg/ml streptomycin, 5 µl of 0.25 mg/ ml amphotericin and 10 µM TSA was added to the cells. In 3D group, the fibrin scaffold was incubated at standard conditions in 6-well plate for 7 days and then 2000 µl cardiogenic medium and 200 µl tranexamic acid were added into a single well and incubated in incubator at 37 °C in a humidified atmosphere containing 5% CO 2 . For both groups after 72 h, the medium was replaced by DMEM/F12 supplemented with 10% FBS and kept for 1, 2, 3, 4 weeks. To check for cell growth and morphological changes in 2D group, they were observed daily by inverted microscope, but the opacity of the scaffold did not allow the viewing of encapsulated cell in 3D by this type of microscope.

Analysis of cardiogenic gene expressions by qRT-PCR
At weeks 1, 2, 3 and 4 of induction total RNA was extracted from treated and untreated hADSCs samples in 2D and 3D groups. RNA extraction was performed by precipitation method (RIBO-Prep, ILS) according to the manufacturer's Instructions. After checking RNA integrity by denaturing agarose gel electrophoresis and ethidium bromide staining, cDNA was synthesized by Revert Aid (Thermo Scientific, USA). Quantitative PCR reactions were performed in duplicate on each sample of cDNA.
Aforementioned reactions were accomplished Maxima SYBR Green qPCR Master Mix (Fermentase, USA) in the presence of forward and reverse primers. SYBR green PCR amplifications were initiated at 95 °C for 10 min followed by 35 cycles of 95 °C, 15 s for denaturation; and 60 °C, 30 s for Annealing/extension. We aimed to analyze quantitative PCR on each sample by Rotor Gene 6000 machine (Qiagen, UK) for NKX2.5, Cx43, cTnI, GATA4, HAND1, HAND2, βMHC and MLC2v genes. Levels of each target gene expression were normalized to the expression of GAPDH (as an internal control) and calculated by the ΔΔCt method.

Analysis of NKX2.5 and cTnI by immunocytochemistry
In the 2D group, the induced cells were fixed with 4% paraformaldehyde while in the 3D group, the scaffolds were fixed in 10% neutral buffered formalin for 12 h at room temperature and paraffin block and then sectioned (thickness 5 µm). Then, the fixed cells in 2 groups were incubated with 0.5% Triton X-100 containing PBS and 10% goat serum for 45 min at room temperature. The protocol was followed by a final incubation with NKX2.5 (1:100; Biorbyte, UK) and cTnI (1:100; Abcam, USA) at 4 °C for 24 h. The next day, the cells were washed and incubated with goat anti-mouse IgG (ab150113, Abcam) and goat anti-rabbit IgG (ab150077, Abcam) at room temperature for 45 min. Then, the cell nucleus was stained by 5 µg/ml of Hoescht 33258 for 10 min. The images were taken under a fluorescence-inverted microscope equipped with a digital camera (DP71, Olympus, Japan).

Statistical analysis
Data from the different tests are presented as mean ± SD of measurements in triplicate and were analyzed by T test and one-way ANOVA followed by post hoc Tukey's test, after normality assumption. P < 0.05 was considered to be statistically significant.
The presence of lipid droplets stained with Oil red O was observed in differentiated hADSCs (Fig. 1a). Alizarin red staining demonstrated calcium phosphate deposits in extracellular matrix of differentiated hADSCs (Fig. 1d). Chondrogenic differentiation of hADSCs was seen by Alcian blue staining (Fig. 1g). No spontaneous differentiation was observed in negative control cultures (Fig. 1c, f, i).

Scanning electron microscopy (SEM)
Structure of the fibrin scaffold was appraised by SEM. A uniform structure of the scaffold with mean pore size of 113.14 ± 26 µm is shown in Fig. 2a.

Cell cycle analysis
Seven days after initial culture, cell cycle analysis was done in two groups. Nearly 44.29% of 2D and 45.12% of 3D cells entered the G0/G1 phase. Moreover, 46.5% of 2D and

Cell morphology changes
Morphological changes of the induced hADSCs with uninduced cells (negative control) during 4 weeks are compared in Fig. 3. The fibroblast-like morphology was the dominant form in negative control group. Gradual morphological changes of the induced hADSCs in the 2D group were multi nuclear, elongated, ball like, myotube-like structures and fork like, while they were star-like during the 4th week. Oval-like and round cell shapes were observed in margin and middle areas of fibrin scaffold. Despite the vast morphological   (Fig. 3).

Analysis of cardiogenic genes by qRT-PCR
In Table 1, final sequences of forward and reverse primers of desired genes are shown. QRT-PCR data are presented in Fig. 4a. Nearly all these genes had higher expression level in 3D group compared with 2D group. Also, differentiation was significantly   3D (c1, c2 and c3) and 2D (b1, b2 and b3) groups. Human heart stained as the positive control (a1, a2 and a3). Cell nuclei (blue) and cardiac Troponin I protein in the nucleus (green) stained by Hoechst overexpressed in some of the genes in 2 groups. Cx43, cTnI, and MLC2v had the same expression patterns with an upward trend from the first week to the 4th week in two groups. In contrast, NKX2.5, HAND1, HAND2 and β-MHC had a downtrend from the first week to the 4th week in two groups. GATA4 had different pattern from the other genes as it had a fast upward trend from the first week to the second week, then it had a slow downtrend from the second week to the 4th week. In addition, NKX2.5 had statistically significant increase during the 1st and 2nd week while GATA4 had the same trend as this increase in the 2nd and 3rd week in 3D group. Also, β-MHC gene showed a significant decrease in expression in 3rd week in 3D culture (Fig. 4a).

Discussion
The techniques for the induction of adult stem cells into cardiac cells need to be refined, since none of them couldn't lead to the creation of functional cardiac cells [20]. Epigenetic modifications have attracted attention in recent years for producing cardiomyocytes from HDACs that have good potential for cardiac differentiation [21]. These epigenetic signatures such as methylation and acetylation on DNA bases or histone proteins have critical roles during the development and differentiation of cells [22]. Histone deacetylases (HDACs) are part of a vast family of enzymes that control cardiovascular growth, development and function [23]. HDACs adjust gene expression patterns by changing chromatin structure. Also, they deacetylate lysine residues and enforce compression of chromatin, decrease the access of transcription factors that are close to the promoters and finally cause gene silencing [24]. TSA, like most HDAC inhibitors, equally inhibits all known HDACs in a reversible fashion by displacing the requisite zinc ion within the active site [25] and removing genes from silent mode [24]. Also, in the process of cardiac cell differentiation, DNA methylation is important in preserving pluripotency and self-renewal of stem cells [17]. 5-Aza by inserting into DNA to form covalent bonds with DNA methyltransferase could inhibit methylation enzyme activity. It has been reported that this hypomethylation, after cell division, can reactivate transcription of genes previously silenced [7]. But, methylation levels from adipose tissue may be theoretically influenced by infiltrated cells, since adipose tissue samples are heterogeneous samples. Also, dynamic alterations in DNA methylation are related to the individual's metabolic state such as; age, sex [26] obesity, caloric restriction, weight loss intervention, exercise, fat distribution and glucose homeostasis [27]. There is even an idea that human adipose mesenchymal cells are not methylated, so using 5-Aza for cardiac differentiation of adipose cells is accompanied with several limitations [17].
Although the cardiac-specific proteins can be expressed with mesenchymal stem cells (MSC) treated with 5-Aza, it seems that 5-Aza cannot be directly involved in stem cell differentiation. However, Wan Safwani et al. [28] believed that the effect of 5-Aza in cardiac differentiation may not be specific [28]. Consequently, effectiveness of 5-Aza should be experimentally determined for particular cells because this effectiveness depends on the hypomethylating level of the cells. Choi et al. [16] only those who compared 5-Aza with TSA on differentiation of hADSCs into cardiac cells. They failed to reproduce cardiac differentiation from hADSCs using 5-Aza until 3nd week while, TSA treatment increased expression level of some cardiac genes 11-fold in comparison with negative control in the 1nd week [16]. However, time is an important factor for cardiac differentiation. In addition, almost all of the studies that used TSA concluded that it was more effective than the other materials for cardiac differentiation [29][30][31].
In process of repair in the heart after an injury, in addition to need a suitable inducer, keeping the injected cells in the damaged area is important issue due to usually more than 94% of the transplanted cells leave the heart. So, using a compatible scaffold is the best way for overcoming this problem. Fibrin is a non-globular protein residing in plasma as fibrinogen precursors which can be polymerized by proteolytic activity of thrombin and makes a fibrin scaffold. If fibrin scaffold combined with mesenchymal cells can be completed the final outcome. Due to, fibrin scaffold have albumin, factor XIII, fibronectin and growth factors, especially platelet-derived growth factors that can be synthesized the matrix [32][33][34]. Also, within the fibrin network there is the arginineglycine-aspartic acid (RGD) motifs that allows cell attachment, increased cell survival and binding of growth factors [32,33]. This scaffold fabricated by fibrin proteins prepares an extracellular matrix like context in which embedded cells are able to simulate real ECM and tissue conditions [35].
Evaluation of the size of the scaffold pores is also optimal for each scaffold system. The pore sizes of the scaffold control its diffusion by transporting nutrients, removing waste, facilitating proliferation and migration of cells and determining the final mechanical properties of the scaffold [36,37]. In this regard, the pore diameter of our scaffolds was 10.54 to 199.4 μm with an average size of 113.14 ± 26 μm.
HDAC inhibitors can affect gene expression patterns nearly in all cell types [38]. NKX2.5 which is a key transcription factor and has a basic duty for heart formation was expressed at the highest level in 2D and 3D groups in the first week. Expression of NKX2.5 represents immature cardiocytes. HAND1 also had the same pattern. It is suggested that HAND1 acts downstream of NKX2.5 to control left ventricular development. At the next rank, GATA4, as early cardiac related gene marker, is expressed at its highest level in the second week. Cardiac contractile proteins (cTnI) and the gap junction protein (connexin43) are expressed at the highest level in the 4th week while another contractile protein, βMHC is expressed at the highest level in the second week. However, the amount of these three genes were not considerable. HAND2 that was expressed with endocardium and epicardium mostly in the primitive right ventricular segment had its highest expression in the second week and after that had a downtrend that might be due to inappropriate and unspecific conditions of cultures.
In vivo studies have shown that there are several stages after an injury occurs to the heart. A blastema that includes cells and expresses early myocardial markers like nkx2.5 and hand2 is first formed. These markers are known as myocardial precursors and begin expression at 3-4 days after ventricular injury. In the second stage, developmental markers are induced and expanded by epicedial tissue and new epithelial coverage is created for the exposed myocardium. A subpopulation of epicardial cells undergoes epithelialto-mesenchymal transition (EMT), makes inroads into the injury location and provides new vasculature to the regenerating muscle [39,40]. Many signaling pathways promote cardiomyocyte proliferation, endocardium activation and epicardial EMT [40].
However, in the present study, MLC2 V known as the structural gene in ventricular development had an expression pattern as cTnI. Overall, our results confirm that the first stage of cardiomyogenesis involves the formation of cardiomyoblasts, which express a clear subset of transcription factors, including Nkx2.5 and GATA4. These genes are involved in activating the expression of the other specific genes to form the differentiated cardiomyocyte [41].

Conclusions
Taken together, we show that TSA helps in fibrin scaffold stimulated differentiation of hADSCs towards cardiac cells during 4 weeks of the experiment. All of the genes were over expressed in the scaffold. This indicates that the fibrin scaffold by secretion of growth factor and other effective factors accompanying human adipose-derived stem cells have high potential for cardiac differentiation and could enhance the inductive abilities of TSA. However, to confirm this content, it is necessary for different dosages of TSA to be investigated in future.


extfieldsofstudy: Biology Medicine

text:
The Evolution, Functions and Applications of the Breast Cancer Genes BRCA1 and BRCA2.

BRCA1 and BRCA2 are both tumor suppressors whose mutations are the cause of most hereditary breast cancers. Both genes are highly involved in ensuring genome stability. BRCA1 homologs are found in the plant and animal kingdoms while BRCA2 homologs are additionally found in the fungi kingdom. The initial origin of both genes remains unknown, however it is expected that the common ancestors originated around 1.6 billion years ago prior to the kingdoms diverging. There has been a great amount of divergence between homologs that is not observed in other tumor suppressors with only functionally important domains conserved. This divergence continues today with evidence of primate BRCA1/2 evolution. Cancer-associated mutations have been found to occur at conserved sites, indicating that conserved sites are important for function. In this study, we present a review on the phylogenesis of BRCA1 and BRCA2.

Abstract. BRCA1 and BRCA2 are both tumor suppressors whose mutations are the cause of most hereditary breast cancers. Both genes are highly involved in ensuring genome stability. BRCA1 homologs are found in the plant and animal kingdoms while BRCA2 homologs are additionally found in the fungi kingdom. The initial origin of both genes remains unknown, however it is expected that the common ancestors originated around 1.6 billion years ago prior to the kingdoms diverging. There has been a great amount of divergence between homologs that is not observed in other tumor suppressors with only functionally important domains conserved. This divergence continues today with evidence of primate BRCA1/2 evolution. Cancer-associated mutations have been found to occur at conserved sites, indicating that conserved sites are important for function. In this study, we present a review on the phylogenesis of BRCA1 and BRCA2.
Breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) are found in a wide variety of organisms and help stabilize the genome. BRCA2 homologs and orthologs are found in organisms across three kingdoms: animal, plant, and fungi whereas BRCA1 homologs and orthologs are only found in animal and plant kingdoms. While not every organism possesses either or both of the genes, a vast majority do, indicating that the presence of BRCA1/2 genes dates back to 1.6 billion years ago when the three kingdoms were first diverging.
Both genes are tumor suppressors and are highly involved in ensuring genome stability. BRCA1 is involved in both the checkpoint activation and in DNA repair, while BRCA2 regulates homologous recombination. BRCA1 and BRCA2 mutations are the cause of most hereditary breast cancer, and are responsible for up to 10% of all breast cancer. The risk for developing breast cancer is 80% with BRCA1 mutations and 60% with BRCA2 (1). While BRCA1 and BRCA2 are notoriously linked to an elevated risk of breast cancer, it has been found that they are also associated with an elevated risk for ovarian, pancreatic, endometrial, prostate, stomach, laryngeal, and fallopian tube cancers (2,3). Breast cancer has been reported in other mammals including rodents, carnivores, and primates (4). This would suggest that BRCA1/2 mutations would affect functions that are unique to mammary tissue and mammals in general, however, that is not the case. The function of both of these genes results in genome stability. BRCA1/2 are both required for homologous recombination. BRCA1 is also involved in cell cycle checkpoints.
Through examining BRCA1/2 in other organisms, the functional domains can be identified that assist in determining if mutations are cancer-causing. Additionally, this allows us to learn more about the function of these genes and possible cures and treatments. Years of research and investigation of various organisms for BRCA1/2 homologs has contributed to our knowledge of BRCA1/2's role in cancer and the evolution of these genes (Table I).

BRCA1 and BRCA2 Function
The initial search for a gene responsible for hereditary breast cancer resulted in finding BRCA1 that is responsible for DNA repair and checkpoints in the cell cycle (5). Further inquires into genes susceptible to breast cancer found BRCA2 which has a role in homologous recombination (6). It has been found that homozygous mutations in either of these genes is lethal and development stops embryonically. Both BRCA1 and BRCA2 lead high-fidelity repair through homologous recombination (7).
The BRCA1 protein exists as a heterodimer with BARD1 and in humans, forms three different complexes. Complex A is involved in DNA repair via homologous recombination, complex B plays a role in the G 1 /S cell-cycle checkpoint, and complex C occurs in the G 2 /M cell-cycle checkpoint (8).
The BRCA2 protein primarily functions in homologous recombination. It is responsible for recruiting RAD51 that is essential for repair. BRCA2 is thought to have a special affinity for ssDNA-dsDNA junctions which are important for recombinational repair in normal replication (9). Additionally, it binds to DSS1 which assists in loading RAD51. BRCA2 also has domains that bind to both ssDNA and dsDNA (10). The main role of BRCA2 in homologous recombination is to load RAD51 onto ssDNA.

Domain Structures of BRCA1 and BRCA2
The human BRCA1 protein has four major domains: RING, P300/CBP, coiled-coil, and BRCT. The RING domain interacts with BARD1 and also shows E3 ligase activity. This activity is amplified when BARD1 and BRCA1 form a heterodimer as it can ubiquitinate itself and enhance its activity (3). The P300/CBP domain has transcriptional regulatory activity. It interacts with two transcriptional cofactors: P300 (histone acetyl transferase p300) and CBP (CREB binding protein) (8). The coiled-coil domain functions in homologous recombination. The domain binds to the coiled-coil domain of PALB2 which then binds the N-terminus of BRCA2 (10). This entire complex then assists with the binding of RAD51 and continuation of homologous recombination. The BRCT domains (BRCA1 carboxy-terminal repeats) are conserved in multiple proteins that are involved in DNA repair. The BRCT domains recognize a phosphorylated serine in the pSXXF motif and bind to three different proteins: Abraxas, BRIP1, and CtIP (8). These different proteins correspond to the complexes A, B, and C respectively. Additionally, the BRCT domains can only bind one protein at a time, indicating that the complexes are mutually exclusive.
The human BRCA2 protein also has four domains: Nterminus, BRC, DBD, and C-terminus. As previously mentioned, the N-terminus binds to PALB2 which assists in the localization of RAD51 to the damaged DNA. The BRC repeats bind to RAD51 and assist in regulating the RAD51 filament formation (3). The DBD (DNA binding domain) contains a helix-rich region, three oligonucleotide/ oligosaccharide binding (OB) fold modules, and a tower domain (TD). Each of these regions have individual functions: OB1, OB2, and OB3 all bind ssDNA, the TD binds dsDNA, and the helix-rich region, OB1, and OB2 bind DSS1 (10). DSS1 is linked to stabilizing BRCA2, promoting homologous recombination, and has many other functions in the cell including DNA repair, development, and protein degradation (8).

Phylogenesis of BRCA1 and BRCA2
The initial origin of both BRCA1 and BRCA2 is still unknown. Both are found only in Eukaryotes; BRCA1/2 are present in plants and animals, and BRCA2 is also found in fungi. Since BRCA2 is found in all three kingdoms, it is likely that the common ancestor of the gene originated prior to the kingdoms splitting around 1.6 billion years ago (11). The common ancestor of BRCA1 also arose around 1.6 billion years ago when plants and animals where diverging. In order to narrow down when either gene arose, more genomes need to be annotated and examined for the functional domains in BRCA1/2.
While BRCA1 homologs have been found in many eukaryotic cells, there is no recognizable one in S. cerevisiae despite the presence of some BRCA1 interacting proteins such as DSS1 (12). BRCA1 has not been identified in any other kingdom thus far, indicating that the common ancestor must be the last common ancestor of plants and animals. All plant BRCA1 homologs except green algae C. reinhardtii and V. carteri have a conserved domain, PHD, that is not present within any animal BRCA1. This suggests that there was a common BRCA1-like gene prior to plant-animal divergence that gained the PHD domain after the split. This hypothesis is confirmed by a paralog of plant BRCA1, BARD1, which also had the PHD domain. In both plants and animals, BARD1 interacts with BRCA1 to form a heterodimer which is necessary for proper BRCA1 function. The plant homolog gained the PHD domain 1.15 billion years ago and the duplication to form BARD1 occurred between 495.9-301 million years ago (8).
BRCA1 homologs have been found in all Viridiplanta genomes except Physcomitrella patens and in most angiosperms (8). Arabidopsis has a BRCA1 homolog (AtBRCA1) that has three of the human domains: RING, BRCT, and P300/CBP (9). Throughout the entire amino acid sequence there is 20% identity between the human and Arabidopsis BRCA1, however, the identity is higher in the domains (8). As previously described, AtBRCA1 also contains the PHD domain between the P300/CBP and the BRCT domains. The function of PHD is still unknown but it is thought to be involved in the regulation of transcription (8).
A BRCA1 ortholog in C. elegans, brc-1, also functions in DNA repair and is found to be elevated during the DNA damage response (13). Overall, the function of BRCA1 and its homologs have not diverged and still maintain a role in genome stability and DNA repair.
BRCA1 homologs are not highly conserved, even amongst closely related species. Entire sequences have low conservation, however, some domains have higher levels of conservation. In BRCA1, a single exon, 11, encodes around 60% of the protein. This exon sequence is more viable than the entire gene for most organisms. In exon 11 within mammals, only 7.95% of the amino acid sequence is fixed and 22.4% is conserved (4). Within exon 11, there are 13 highly conserved regions between eutherian mammals, chicken, frog, and marsupials; three of these regions are unique to mammals (4). Since the divergence of primates from non-primates 74.1 million years ago, 132 amino acid sites in the human BRCA1 have evolved (14,15).
BRCA1 homologs do not share the high degree of similarity that is implied by the significant function of the gene. There is only 56% identity between the human and mouse BRCA1. Compared to other genes, this level of identity is low and displays the poor sequence conservation. MSH2 and XPA, both tumor suppressor genes, have a 92% and 86% identity, respectively, between human and mouse sequences. RAD51, which interacts with BRCA2 and is in the BRCA1 complex A, has a 99% identity between human and mouse (15). Despite the low levels of conservation, it appears that function of BRCA1 has not evolved.
Similarly to BRCA1, there is no BRCA2 homolog in S. cerevisiae, however, there are homologs in other fungi. In U. maydis, the BRCA2 homolog is Brh2 and interacts with RAD51. Brh2 requires BRC elements in order to interact with RAD51. There is only one BRC domain in Brh2 whereas there are 8 BRCs in the human BRCA2. Brh2 has no overall sequence alignment with any known gene, however, within the sequence are regions that align with BRCA2 conserved domains. The ability of BRCA2 homologs to associate with RAD51 is conserved in U. maydis, Arabidopsis, C. elegans, and all the way up to mammals (15). In U. maydis, Brh2 functions as a master regulator of recombinational machinery. Brh2 is required for proficiency in repair and recombination and serves in maintaining genome stability in mitosis and meiosis (18). This function is more extensive than the human BRCA2 function suggesting that the function has narrowed and become more specified through evolution. This sequence has conservation with homologs in Arabidopsis and Oryza, with 38% identity in the C terminus and the nuclear localization signal (NLS). The residues that directly interact with DSS1 are the most highly conserved indicating that the interaction between them has a biologically important function (19).
Arabidopsis the is only known organism to have two BRCA2 homologs. They are 96.8% identical to each other which suggests that one arose from the other through a duplication. There is 21% identity to human BRCA2 (9). The function of these homologs has been found to be essential in meiosis. Plant homologs for BRCA2 have been found in Viridiplantae and in green algae as well. Unlike animals, plants that either lack BRCA2 or are homozygous mutants are able to survive past embryonic development (8).
The chicken BRCA2 homolog has the same order and organization as the mammalian version, however, it is poorly conserved with only 37% amino acid identity. Exons 2, 4, 7, and 16-20 are highly conserved between the two homologs with an average identity of 70% (20). Any high conservation within a poorly conserved gene indicates important

Authors
Year Impact Kojic M et al. (18). 2002 BRCA2 homolog was found in Ustilago maydis, a fungus, that interacts with the Rad51 ortholog in the same manner as in humans. Prior to this discovery, it was believed that only higher mammals possessed BRCA2 homologs. This indicated that BRCA2 was widespread across multiple kingdoms. Fleming MA et al. (15). 2003 Conserved regions were identified between mammalian BRCA1 homologs. The conserved regions are all located in domains that are known to interact with other proteins. Three regions were identified as being under positive selection. This suggested that the BRCA1 gene is still evolving and that there is a force that is driving the selection. Ramirez CJ et al. (4). 2004 Conserved regions were identified in exon 11 of animal BRCA1 homologs. Missense changes that are likely to disrupt gene function were identified and over half of those identified occurred in highly conserved regions of the gene. This indicates the importance of finding homologs and the conserved regions of genes as it can help identify mutations that disrupt function and are likely disease causing. Dray E et al. (17). 2006 The Arabidopsis ortholog of BRCA2 was shown to interact with orthologs of the proteins the human BRCA2 does. The specific domains that are necessary to interact were also determined. These domains can be used to evaluate possible disease causing mutations. Lou DI et al. (1). 2014 In primates, BRCA1 and BRCA2 are evolving through positive selection. The hypothesis for this evolution is that viral infections are driving the selection. The mutations in BRCA1 and BRCA1 make the cell less susceptible to viral infection. This results in antagonistic pleiotropy. This hypothesis explains why BRCA1/2 continue to evolve and pass on cancerous mutations. Zhou Q et al. (19). 2014 The DNA binding domains of the BRCA2 ortholog in Ustilago maydis were identified and found to be similar to one another yet are involved in different aspects of the repair process. Continuing to determine the function of every domain of BRCA2 leads to more insights on the effects of mutations.
functionality. Exons 2 and 4 are in the N-terminal domain and exons [16][17][18][19][20] are in the C-terminal domain which supports BRCA2 conserving function. The largest exon, 11, harbors the BRC repeats which are responsible for binding RAD51. In chicken BRCA2, exon 11 is poorly conserved with 21% identity to human BRCA2. However, three of the BRC repeats have a 70% identity between the homologs (20). The BRC repeats are highly variable among all BRCA2 homologs. The repeats range from 1 repeat to 15 repeats in some species. The duplication of the BRC repeats happened around 230-300 million years ago (20).
Just as the BRCA1 homologs are poorly conserved, the BRCA2 homologs follow this same pattern. The highest conserved regions across all homologs are the BRC repeats and the C-terminal region (18). The divergence amongst the homologs could reflect the diversification and specialization in the regulation of RAD51-mediated DNA repair throughout the evolutionary tree.

Human BRCA1/2 Genes
The human BRCA1 gene is located at chromosome 17q21.3. The entire gene is 193,689 nucleotides; the coding sequence begins in the second exon and ends in the 24th exon. Exon 11 encodes more than 60% of the entire protein (21). The protein is 1,863 amino acids long and 220 kDa. The BRCA2 gene is located at chromosome 12q13.1. The entire gene is 91,193 nucleotides and the coding region begins in the first exon and ends in the 27th exon (22). Exon 11 encodes more than 50% of the entire protein. The BRCA2 protein is 3,418 amino acids long and 390 kDa.

Limitations of Evolutionary Studies
When looking the evolutionary relationship of BRCA1/2, we rely on the published genomes. However, the accuracy of these genomes cannot be confirmed. Many genomes are likely to have poor assembly of the genome, or missing a percentage of the genes. Additionally, since the history of both BRCA1 and BRCA2 extends over one billion years, only domains or residues that are continuously selected for are truly conserved. This can lead to difficulty establishing the origin of the gene because so much divergence has occurred and can also lead to genes being included that are not true homologs, but instead just have similar domains and functions. Therefore, conclusions about the evolutionary development of either BRCA1 or BRCA2 must be taken as good estimates of past events, not as definitive statements.

Relationship Between BRCA1 and BRCA2
There is no phylogenetic relationship between BRCA1 and BRCA2. Although BRCA1 and BRCA2 have similar disease phenotypes and both play a role in homologous recombination, there is no homology between each other. Neither gene shows homology to other genes in the human genome (3). Both genes are thought to have the same disease phenotype because of their joint role in homologous recombination.

Continued Evolution of BRCA1 and BRCA2
There is evidence that both BRCA1 and BRCA2 are still evolving, particularly in primates. It is unknown as to why both of these genes are continuing to evolve as rapidly as they are.
There are indications that there is a selection occurring at specific loci in the modern day (1). There have been 22 nonsynonymous mutations in the human BRCA1 gene since the divergence from chimpanzees and bonobos six million years ago. BRCA1 has far more non-synonymous mutations than other genes, indicating that it is evolving and is occurring much faster than expected. Two SNPs in the chimpanzee BRCA1 were found to not be in Hardy-Weinberg equilibrium. This suggests that more than just the human BRCA1 gene is evolving. In simian primates, there has been a selection that favors non-synonymous mutations with BRCA1 that has lasted through the speciation of the primates. The most intense selection found has occurred in the human/chimpanzee/bonobo clade (1). Most of the selection observed is positive selection. Evidence for this rapid evolution within the human BRCA1 gene along with other primate homologs continues to be found across the gene (1,4,15). The evolution of BRCA1 in humans in occurring rapidly despite the fact that many of the changes to the gene result in a significantly elevated risk for breast cancer. Despite the rapid evolution in primate BRCA1, there has been no identification of rapid evolution in marsupial BRCA1 homologs. This indicates that the selection force is acting only upon primates.
BRCA2 is also evolving under positive selection only in primates. There have been three sites in exon 11 of the human BRCA2 that are under positive selection in the RAD51 interacting domain (15). The selection in BRCA2 is not concentrated in the human/chimpanzee/bonobo clade as was seen in BRCA1. This selection is not as strong as the selection observed in BRCA1, but it is still occurring more rapidly than typically predicted. It remains unknown as to why these genes are evolving at this elevated rate.
The strongest hypothesis for this phenomenon is viral infections are driving this rapid evolution. Many viruses interact with DNA repair proteins in order to infect the host and remain undetected. The introduction of viral DNA can trigger the DNA damage response, yet in order to effectively infect, the virus must impede this response (23). Some viruses also use the DNA repair proteins in order to replicate themselves. These viruses could be interacting with either BRCA1 or BRCA2 and driving the changes within the genes to become less susceptible to these viruses. This selection leads to decrease vulnerability to infection, however, it also leads to consequences for the host. This scenario could lead to antagonistic pleiotropy where BRCA1/2 are less susceptible to viral infection but increase the risk of developing breast cancer (1).

Role of BRCA1 and BRCA2 in Cancer
Many of the inherited BRCA1 mutations that are cancerassociated have been found within the BRCT and RING domains which indicates that those domains are essential to the function of BRCA1 (3). There have been 38 missense changes in exon 11 of BRCA1 identified that are likely to have an effect on protein function (15). Of these 38, 3 effect fixed locations and 3 affect conservative sites as determined by the analysis of mammalian BRCA1 homologs. Since the mutations affect fixed or conservative sites, they are more likely to be cancer-associated mutations as they disrupt loci that have an essential role in the protein function. The knowledge of conservative locations can be used to determine if variants are simply polymorphisms or disease associated variants. If the variant occurs at a fully or partially conserved residue, then the variant is probably diseaseassociated. Variants that have no disease link or polymorphisms typically occur in non-conserved regions.
Although mutations in both BRCA1 and BRCA2 increase the risk for breast cancer, they cause different cancer types. BRCA1 mutations typically result in triple-negative breast cancer (TNBC) where there is no overexpression of estrogen receptor (ER), progesterone receptor, or human epidermal growth factor receptor-2 (2). This cancer type is characterized by aggressive growth and very few targeted treatment options. The desired treatment for TNBC would only target cells with the BRCA1 mutation and not healthy cells, however, since there is no overexpression, there is no target for treatment. The use of poly ADP-ribose polymerase-1 (PARP1) inhibitor and gemcitabine has shown to be a promising treatment and prompts BRCA1 mutated cells to go through apoptosis (24). Conversely, BRCA2 mutations result in low-grade ER + luminal tumors that grow more slowly and have the treatment option of inhibiting signaling through ER (2).
While BRCA1/2 mutations also elevate the risk of many different types of cancer, breast cancer remains the most prominent. It is still not fully understood as to why these mutations impact mammary tissue in particular, especially considering the broad role of genome stabilization that genes carry. BRCA1 has been found to be a transcriptional regulator of ER; BRCA1 acts to repress the estrogen receptor which prevents further signaling that promotes cell proliferation (25). If BRCA1 is mutated and cannot act as a regulator, then the ER can over promote cell proliferation.
Another hypothesis is that the hormonally driven growth during the menstrual cycle produces reactive oxygen species which causes DNA damage (3). This DNA damage paired with a nonfunctional BRCA1 or BRCA2 leads to increased genome instability and more DNA mutations. In a study by Malicka et al. (26), it was found that physical therapy in rats lead to significantly higher progesterone levels that was found to inhibit mammary gland cancer and increase tumor cell apoptosis. This suggests that hormone treatment or physical therapy could have a suppressive effect on breast cancer development (26).
The common link between BRCA1 and BRCA2 is homologous recombination. Since BRCA1 and BRCA2 mutations have the same disease phenotype, it can be concluded that homologous recombination is the critical system for protecting the genome. It is this pathway that is affected in both mutations, confirming that the role of BRCA1/2 in homologous recombination is essential for tumor suppression.

Conclusion
Both the tumor suppressor genes BRCA1 and BRCA2 have been around for approximately 1.6 billion years in animals and plants, with BRCA2 in fungi as well. Much of our knowledge about both of these genes have come from studying homologs in various organisms. Additionally, the realization that both BRCA1 and BRCA2 are still rapidly evolving in primates explains the perseverance of disease causing mutations in the genes.

Conflicts of Interest
The Authors declare no competing financial interests.