**Chapter 2**

[35] Chakrapani Y, Soundararajan K. Genetic algorithm applied to fractal image compression. ARPN Journal of Engineering and Applied Sciences.

*Fractal Analysis - Selected Examples*

International Journal of Engineering Research & Technology. 2012;**1**(10):1-5

[43] Wang X-Y, Zhang D-D, Wei N. Fractal image coding algorithm using particle swarm optimization and hybrid Quadtree partition scheme. IET Image

[44] Wang X-Y, Zhang D-D. Discrete wavelet transform-based simple range classification strategies for fractal image coding. Nonlinear Dynamics. 2014;

Processing. 2015;**9**(2):153-161

**75**(3):439-448

[36] Xing-yuan W, Fan-ping L, Shu-guo W. Fractal image compression based on spatial correlation and hybrid genetic

Representation. 2009;**20**(8):505-510. DOI: 10.1016/j.jvcir.2009.07.002

[37] Chakrapani Y, Soundararajan K. Implementation of fractal image compression employing particle swarm optimization. The World Journal of Modelling and Simulation. 2010;**6**(1):

[38] Al-Bundi SS, Al-Saidi NMG, Al-Jawari NJ. Crowding optimization method to improve fractal image compressions based iterated function systems. International Journal of Advanced Computer Science and Applications. 2016;**7**(7):392-401

[39] Al-Saidi NMG, Al-Bundi SS, Al-Jawari

[40] Menassel R, Nini B, Mekhaznia T. An improved fractal image compression using wolf pack algorithm. Journal of Experimental & Theoretical Artificial Intelligence. 2018;**30**(3):429-439. DOI: 10.1080/0952813X.2017.1409281

[41] Menassel R, Gaba I, Titi K.

1206212X.2019.1638631

**20**

optimization of fractal image

Introducing BAT inspired algorithm to improve fractal image compression. International Journal of Computers and Applications. 2019. DOI: 10.1080/

[42] Kalunge VV, Varunakshi B. Time

compression by using genetic algorithm.

NJ. An improved harmony search algorithm for reducing computational time of fractal image coding. Journal of Theoretical and Applied Information Technology. 2017;**95**(8):1669-1678

algorithm. Journal of Visual Communication and Image

2009;**4**(1):53-58

40-46


Tribological Performance of Random Sinter Pores vs. Deterministic Laser Surface Textures: An Experimental and Machine Learning Approach

*Guido Boidi, Philipp G. Grützmacher, Markus Varga, Márcio Rodrigues da Silva, Carsten Gachot, Daniele Dini, Francisco J. Profito and Izabel F. Machado*

## **Abstract**

This work critically scrutinizes and compares the tribological performance of randomly distributed surface pores in sintered materials and precisely tailored laser textures produced by different laser surface texturing techniques. The pore distributions and dimensions were modified by changing the sintering parameters, while the topological features of the laser textures were varied by changing the laser sources and structuring parameters. Ball-on-disc tribological experiments were carried out under lubricated combined sliding-rolling conditions. Film thickness was measured *in-situ* through a specific interferometry technique developed for the study of rough surfaces. Furthermore, a machine learning approach based on the radial basis function method was proposed to predict the frictional behavior of contact interfaces with surface irregularities. The main results show that both sintered and laser textured materials can reduce friction compared to the untextured material under certain operating conditions. Moreover, the machine learning model was shown to predict results with satisfactory accuracy. It was also found that the performance of sintered materials could lead to similar improvements as achieved by textured surfaces, even if surface pores are randomly distributed and not precisely controlled.

**Keywords:** lubrication, friction reduction, laser surface texturing, sintered material, machine learning

#### **1. Introduction**

The surface topography of mechanical components is often modified to improve their tribological performance, such as cylinder liner honing in internal combustion engines [1] and laser surface texturing in piston-rings [2, 3], rolling element bearings [4], and journal- and thrust bearing systems [5].

Surface textures can promote several tribological improvements. Surface cavities can reduce abrasive wear in harsh contact conditions by retaining wear and

contaminant particles (debris trapping effect), as well as work as micro-reservoirs and secondary oil suppliers (oil reservoir effect) [6]. Under mixed and (elasto) hydrodynamic lubrication, friction can be reduced by the interplay of different mechanisms, such as the (i) micro-hydrodynamic bearing (boosting in the fluid pressures due to the texture-induced cavitation at the divergent regions of the textures) [7], (ii) inlet suction (lubricant sucking into the interface due to the difference between the supply pressure and the cavitation pressure) [3, 8, 9], and (iii) shear-area variation (decrease of the fluid and contact shear stresses over the contact area) [10] mechanisms. Furthermore, under boundary and mixed lubrication regimes, textures can influence the sealing performance and percolation behavior. The reader is referred to the comprehensive reviews [4, 11–14] for a more in-depth evaluation of several aspects of surface texturing for tribological improvements.

The tribological effects of surface texturing were first studied in the 1960s by Hamilton's and Anno's research groups [15, 16]. Afterwards, this topic covered a marginal role in the tribological community until the 1990s, when Etsion and coworkers re-discovered its potential impact [17, 18], also in the context of improved manufacturing techniques. In recent years, the interest in research on surface texturing, mainly laser texturing, has significantly increased [11] as some texture configurations have shown significant tribological improvements in various machine elements [2, 19–21].

The design of effective surface textures requires a thorough understanding of the tribosystem's characteristics and the capabilities and limitations of the texturing techniques available. General guidelines for texture design could be found in [4, 12]. Nevertheless, further in-depth research is still required to reveal the precise mechanisms (and their interplay) responsible for the improvements in tribological performance due to the presence of optimally designed micro-textured surfaces; this is still not fully understood. This lack of understanding is mainly related to the design and manufacturing limitations of optimal texture geometries for different components, the influence of varying operating conditions in transient applications, and the evolution of the texture geometry over the components' lifetime. Additionally, there is a particular debate on the effect of surface textures on the behavior of lubricated non-conformal contacts under different sliding-rolling conditions. Finally, to successfully and economically implement surface texturing in practical applications and industrial scale, the gain in tribological performance must compensate for the additional manufacturing steps leading to longer processing times and costs.

Surface porosity in sintered materials could be potentially used as inherent surface texture and saving extra manufacturing. Pore characteristics can be controlled, to certain limits, by the sintering parameters and powder properties [22]. However, in contrast to deterministic laser textures – with a precisely tailored topography – surface pores are statistically distributed and have irregular shapes. The effect of pores on mechanical proprieties of sintered materials was extensively studied [23–26]; however, the influence of surface porosity on the tribological performance is sparsely investigated [27–29].

Advanced statistics, artificial intelligence (AI) and machine learning (ML) methods have gained increasing importance in describing and interpreting scientific findings. In tribology research, ML & AI approaches have already been used for online condition monitoring of bearings, design of material composition, lubricant formulations, lubrication and fluid film formation analysis, among other applications [30–32]. An ML method was also recently proposed to predict the frictional performance of textured and porous interfaces [33], whose methodology could be extended to support the optimum design of surface texturing.

*Tribological Performance of Random Sinter Pores vs. Deterministic Laser Surface Textures… DOI: http://dx.doi.org/10.5772/intechopen.100245*

This study aims to quantitatively compare random surface features of porous sintered materials with distinctly manufactured laser surface textures in the particular case of lubricated non-conformal contacts. Especially the sliding-rolling conditions as occurring in roller-bearings are tackled. Finally, the radial basis ML method was used to enhance results interpretation and build a general predictive model to support the design of new surface features for obtaining superior tribological performances.


*4.3.5 Biodiversity conservation in Kenya*

Although Kenya's biodiversity remains highly protected, declines are common phenomena due to a number of anthropogenic threats that have led to numerous conservation challenges [57]. Nevertheless, the country explores all avenues to ensure that efforts to win the war against biodiversity losses are sustained. Examples of progress made in research and development for drylands afforestation in Kenya include the following:

#### *<IP_ADDRESS> Selection of appropriate tree species*

Until the 1980s–90s, when it became a government policy in Kenya to promote the replanting of indigenous rather than exotic tree species, most of the work on the selection of trees of arid and semiarid lands in Kenya was with exotic fastgrowing species [53]. Since then, selection criteria have continued to evolve with consideration around preferences of local communities, availability of quality genetic material for propagation, and site biophysical conditions. Currently, a large number of tree species have been recommended for the drylands of Kenya [52, 53]. In the dryland areas of Kitui and Kibwezi (Eastern Kenya), tree species grown and recommended include *Azadirachta indica*, *Jatropha curcas*, *Senna siamea*, *Leucaena leucocephala*, *Croton megalocarpus*, *Casuarina equisetifolia*, *Melia volkensii*, *Eucalyptus camaldulensis*, and *Dovyalis caffra* [4].

In Kenya, the area under *Eucalyptus* is likely to increase as a result of high demand for transmission poles, for construction, fuelwood, carbon sequestration, and mitigation of the effects of climate change [58]. However, there is much unease about Eucalyptus water consumption as compared to other woody flora. On a positive note, studies have established that Eucalypts exhibit high efficiency in water use for biomass accumulation. It has been established that eucalyptus requires less water to produce one (1) kg of biomass than most crops [59].

*Melia volkensii*, an important timber species that grows well in well-drained soils, is a promising indigenous tree species found in the drylands of Kenya. It is fast growing, drought tolerant, and produces high-quality hardwood timber for furniture. However, this species is heavily exploited in its natural stands and the trend has been worsening over the last decade owing to shortage of alternative hardwood species in drylands. As a result, programs promoting domestication of the species as a plantation species are ongoing [60–62].

#### *<IP_ADDRESS> Development of suitable methods for propagation*

Poor propagation of some promising tree species in the drylands of Kenya has slowed down the country's efforts to increase its forest cover to the targeted 10%. For example, lack of seedlings attributed to poor seed germination is experienced with *Terminalia brownii*, a drought-tolerant species, which can be used to rehabilitate degraded drylands through reforestation and agroforestry approaches [63]. The current demand for Terminalia seedlings is higher than the supply. Research has focused on development of technologies ranging from breakage of dormancy to plant-tissue cultures to improve germination propagation of such species. Studies have been conducted to investigate the dormancy and germination of *T. brownii* seeds collected from various dryland sites in Kitui, Makueni, Tharaka-Nithi, and Baringo Counties of Kenya. Extracted seeds recorded the highest germination with the best at 76% compared to nipped seeds (13%) and those subjected to other treatments [63]. *Melia volkensii* Gurke is another drought-tolerant tree native to the drylands, of which cultivation is limited by difficulties in propagation via conventional means. Full exploitation of the ability of thidiazuron to elicit regeneration in plant-tissue cultures, as a sole plant growth regulator, was found to be hampered by high costs. Alternative effective and low-cost agrochemical thidiazuron for in vitro propagation of *M. volkensii* was found to be Kingtai-TDZ, which has a high potency and suitability for use in tissue culture of the species [61]. Because of difficulties in seed germination, land users sometimes go for the use of plants produced from root and stem cuttings, rather than from seedlings, and researchers have focused on the possibility that root and stem cuttings may be used for propagation, rather than seedlings [62]. However, if cuttings

are used to circumvent the problems of seed germination, alternative methods of controlling competition, such as root pruning, need to be considered.


**Does the Number of Retrieved Oocytes Influence Pregnancy Rate After Day 3 and Day 5 Embryo Transfer?**

Veljko Vlaisavljević, Jure Knez and Borut Kovačič *Department of Reproductive Medicine, University Medical Centre Maribor Slovenija*

## **1. Introduction**

38 Advances in Embryo Transfer

[68] Ata B, Yakin K, Balaban B, Urman B: Embryo implantation rates in natural and

[69] Verberg MFG, Macklon NS, Nargund G, Frydman R, Devroey P, Broekmans FJ,

Biomed Med Online 2008;77:207-212.

29.

stimulated assisted reproduction treatment cycles in poor responders. Reprod

Fauser BCJM: Mild ovarian stimulation for IVF. Hum Reprod Update 2009;15:13-

Decisions associated to ovarian stimulation approach are an essential component of medically assisted reproduction (MAR). Considerable amount of research in this field has enhanced our understanding of certain biological phenomena taking place in the process and also brought along novel means of ovarian stimulation. Nevertheless, there is still not enough evidence to suggest the optimal number of oocytes collected at retrieval in order to predict the occurrence of successful pregnancy and most importantly, birth of a live baby. First, this chapter will describe certain historical developments in MAR. Next, current ovarian stimulation protocols and desired aims of modern era ovarian stimulation, embryo culture and embryo transfer outcome will be discussed. A single-centre MAR results analysis was performed in a ten year span in order to help resolve one of the ultimate questions: what is the optimal number of oocytes needed to achieve clinical pregnancy after embryo transfer?

## **2. Milestones in evolution of ovarian stimulation and embryo transfer**

Ever since the beginnings of MAR, increasing the chance of a live birth has been the most important aim of researchers' efforts. The first successful embryo transfers (ETs) resulting in pregnancy by Edwards and Steptoe in the early 1970s were carried out in natural cycles. This means that only one oocyte was harvested at follicle aspiration (which was at the time performed laparoscopically). Hundred-and-one ETs were attempted before the first successful delivery of the world's first IVF (in vitro fertilization) baby Louise Brown in 1978 (Edwards & Steptoe, 1980). In these first attempts that paved the way for future extensive worldwide MAR success, Edwards and Steptoe described 65 natural cycles that yielded 45 oocytes from 44 patients and resulted in 3 successful deliveries. Thus, pregnancy rate was 9.1% and delivery rate 6.8% per oocyte retrieval.

Since then, research has brought along many improvements to MAR. In the early 1980s, one of the most important steps was the introduction of human menopausal gonadotrophin (HMG), which allowed for controlled ovarian hyperstimulation (COH) and multiple follicular growth. This enabled the retrieval of multiple oocytes at pick-up and thus substantially increased the likelihood of successful embryo replacement and subsequent

Does the Number of Retrieved Oocytes Influence

**3.3 Embryo cultivation and transfer strategies**

transfer of cleavage stage embryos (Blake et al., 2007).

transferred at cleavage or blastocyst stage.

Pregnancy Rate After Day 3 and Day 5 Embryo Transfer? 41

procedures. Due to the lack of reliable data, experts' opinions of such legislation effects on treatment success are too often contradictory. Thus, further research is needed to establish

In the scenario of transfer of more than one embryo, multiple gestations are the most common complication of pregnancies achieved through MAR. Thus, in recent times, scientific societies have propagated the idea that the goal of medically assisted procreation must be the achievement of a singleton pregnancy (European Society of Human Reproduction and Embryology [ESHRE] Task Force on Ethics and Law, 2003). The simplest way of reducing multiple gestation incidences is strict implementation of single embryo transfer (SET) strategy. However, this is related to significant declines of pregnancy rates. The development of advanced embryo culture media in the past two decades have allowed for extended, blastocyst cultivation of the embryos. Following the evolution of these media, many have advocated blastocyst transfer mainly due to better morphologic embryo assessment possibilities at blastocyst stage compared to cleavage stage embryos. Although the opinions on this subject are still not uniform, many recently performed studies have demonstrated significantly better outcome after blastocyst transfer (Papanikolau et al., 2008). Thus, a recent Cochrane review has shown a significant improvement in pregnancy and live birth rates for blastocyst transfer compared to

Currently, increasing number of legislation acts on MAR in developed countries impose mandatory single embryo transfer under certain circumstances (mostly in younger patients), in order to decrease the incidence of multiple gestations. Thus, the question remains; how to select the best embryos in order to achieve the highest success and at the same time lower the incidence of multiple gestations? With regards to these facts, additional studies should settle the issue in what scenario can the number of oocytes be a factor in decision for SET or multiple embryo transfer and whether embryos should be

**4. Influence of number of retrieved oocytes on embryo transfer success**

A retrospective study of retrieved oocyte number on MAR outcome was performed at Department of Reproductive Medicine and Gynaecologic Endocrinology, University Medical Centre in Maribor. Totally, 6989 consecutive in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles resulting in follicle aspiration and oocyte pick-up were included. Ovarian stimulation protocols with combination of GnRH agonist/GnRH antagonist and recombinant FSH (Gonal-f®, Serono International SA, Geneva, Switzerland)/HMG (Menopur®, Ferring Pharmaceuticals Inc., Saint-Prex, Switzerland) were used and were previously described in detail (Vlaisavljević et al., 2000). Embryo quality was assessed by an experienced embryologist at day two and blastocysts were graded according to our established grading system at day five after oocyte pick-up (Kovačič et al., 2004). Embryo transfer was carried out three or five days after oocyte pick-up. Day five blastocyst transfer was performed if more than four fertilised oocytes were obtained and if more than three optimal embryos were available on day three according to our standard policies (Kovačič et al., 2002). After consultation with the patients, time of embryo transfer could be adjusted to

the role of number of harvested oocytes in MAR outcome prediction.

pregnancy. This finding has ignited further research and development that has improved gonadotrophin formulation and efficiency, but detailed illustration of this process is beyond the scope of this chapter.

## **3. Current challenges in ovarian stimulation**

#### **3.1 Ovarian hyperstimulation syndrome**

The development and implementation of gonadotrophins that allowed for controlled ovarian hyperstimulation (COH) in MAR has brought along a possibility of potentially lifethreatening complication, namely ovarian hyperstimulation syndrome (OHSS) (Brinsden et al., 1995). It has been established that the risk of OHSS increases proportionally with an increase in ovarian response to stimulation as measured by the number of ovarian follicles, number of retrieved oocytes and serum estradiol concentration on the day of human chorionic gonadotrophin (hCG) administration (Asch et al., 1991). In our previous research, it has been demonstrated that the risk of OHSS increases with rising number of harvested oocytes and the risk is significantly higher in patients with more than ten collected oocytes (Reljič et al., 1999). Severe OHSS is a serious and potentially life-threatening complication of MAR treatment and has a mean incidence of 1-3% in MAR programmes involving standard ovarian stimulation protocols (Fauser et al., 1999). However, to date, there are no reliable predictors of its occurrence. Owing to these facts, many investigations in recent decade have been aimed towards less aggressive, milder forms of ovarian stimulation and to procedures that could eliminate the risk of OHSS (Revelli et al., 2011).

#### **3.2 Ovarian stimulation strategies**

Ovarian stimulation is the crucial component in medically assisted reproduction. In current practice, long acting gonadotrophin-releasing hormone (GnRH) agonist pituitary suppression combined with recombinant or purified urinary exogenous follicle stimulating hormone (FSH) is the most frequently used stimulation protocol (Macklon et al., 2006). However, in light of making MAR more "patient friendly", there is a recent trend toward milder stimulation protocols in order to reduce the chances of complications and not lastly, to lower the costs of MAR treatment (Fauser et al., 1999). With the availability of GnRH antagonists in ovarian stimulation protocols, administration of FSH can be delayed to midfollicular phase, thus reducing the amount of gonadotrophins used for stimulation and minimizing exogenous hormonal interferences that are present in conventional hormonal stimulation (Fauser & van Heusden, 1997). On the other hand, regarding reduction of OHSS, conventional stimulation protocols employing GnRH antagonists allow for substitution of human chorionic gonadotrophin (hCG) with GnRH agonist for ovulation triggering. Using this procedure, OHSS can be eliminated almost completely even in high responding patients with high numbers of retrieved oocytes (Humaidan et al., 2011).

Despite the novel approaches to ovarian stimulation, the number of retrieved oocytes is still considered to be an important prognostic variable in everyday MAR practice. However, the relation between the number of oocytes and MAR outcome is poorly understood as studies performed on this subject present with conflicting results (Hamoda et al., 2010; Letterie et al., 2005; Meniru et al., 1997; Sunkara et al., 2011; Yoldemir et al., 2010). Moreover, as IVF procedures are performed in increasing extent globally, new, restrictive legislation policies in certain countries limit the amount of oocytes per cycle that can be used in MAR

pregnancy. This finding has ignited further research and development that has improved gonadotrophin formulation and efficiency, but detailed illustration of this process is beyond

The development and implementation of gonadotrophins that allowed for controlled ovarian hyperstimulation (COH) in MAR has brought along a possibility of potentially lifethreatening complication, namely ovarian hyperstimulation syndrome (OHSS) (Brinsden et al., 1995). It has been established that the risk of OHSS increases proportionally with an increase in ovarian response to stimulation as measured by the number of ovarian follicles, number of retrieved oocytes and serum estradiol concentration on the day of human chorionic gonadotrophin (hCG) administration (Asch et al., 1991). In our previous research, it has been demonstrated that the risk of OHSS increases with rising number of harvested oocytes and the risk is significantly higher in patients with more than ten collected oocytes (Reljič et al., 1999). Severe OHSS is a serious and potentially life-threatening complication of MAR treatment and has a mean incidence of 1-3% in MAR programmes involving standard ovarian stimulation protocols (Fauser et al., 1999). However, to date, there are no reliable predictors of its occurrence. Owing to these facts, many investigations in recent decade have been aimed towards less aggressive, milder forms of ovarian stimulation and to procedures

Ovarian stimulation is the crucial component in medically assisted reproduction. In current practice, long acting gonadotrophin-releasing hormone (GnRH) agonist pituitary suppression combined with recombinant or purified urinary exogenous follicle stimulating hormone (FSH) is the most frequently used stimulation protocol (Macklon et al., 2006). However, in light of making MAR more "patient friendly", there is a recent trend toward milder stimulation protocols in order to reduce the chances of complications and not lastly, to lower the costs of MAR treatment (Fauser et al., 1999). With the availability of GnRH antagonists in ovarian stimulation protocols, administration of FSH can be delayed to midfollicular phase, thus reducing the amount of gonadotrophins used for stimulation and minimizing exogenous hormonal interferences that are present in conventional hormonal stimulation (Fauser & van Heusden, 1997). On the other hand, regarding reduction of OHSS, conventional stimulation protocols employing GnRH antagonists allow for substitution of human chorionic gonadotrophin (hCG) with GnRH agonist for ovulation triggering. Using this procedure, OHSS can be eliminated almost completely even in high responding patients

Despite the novel approaches to ovarian stimulation, the number of retrieved oocytes is still considered to be an important prognostic variable in everyday MAR practice. However, the relation between the number of oocytes and MAR outcome is poorly understood as studies performed on this subject present with conflicting results (Hamoda et al., 2010; Letterie et al., 2005; Meniru et al., 1997; Sunkara et al., 2011; Yoldemir et al., 2010). Moreover, as IVF procedures are performed in increasing extent globally, new, restrictive legislation policies in certain countries limit the amount of oocytes per cycle that can be used in MAR

the scope of this chapter.

**3. Current challenges in ovarian stimulation**

that could eliminate the risk of OHSS (Revelli et al., 2011).

with high numbers of retrieved oocytes (Humaidan et al., 2011).

**3.2 Ovarian stimulation strategies**

**3.1 Ovarian hyperstimulation syndrome**

procedures. Due to the lack of reliable data, experts' opinions of such legislation effects on treatment success are too often contradictory. Thus, further research is needed to establish the role of number of harvested oocytes in MAR outcome prediction.

## **3.3 Embryo cultivation and transfer strategies**

In the scenario of transfer of more than one embryo, multiple gestations are the most common complication of pregnancies achieved through MAR. Thus, in recent times, scientific societies have propagated the idea that the goal of medically assisted procreation must be the achievement of a singleton pregnancy (European Society of Human Reproduction and Embryology [ESHRE] Task Force on Ethics and Law, 2003). The simplest way of reducing multiple gestation incidences is strict implementation of single embryo transfer (SET) strategy. However, this is related to significant declines of pregnancy rates. The development of advanced embryo culture media in the past two decades have allowed for extended, blastocyst cultivation of the embryos. Following the evolution of these media, many have advocated blastocyst transfer mainly due to better morphologic embryo assessment possibilities at blastocyst stage compared to cleavage stage embryos. Although the opinions on this subject are still not uniform, many recently performed studies have demonstrated significantly better outcome after blastocyst transfer (Papanikolau et al., 2008). Thus, a recent Cochrane review has shown a significant improvement in pregnancy and live birth rates for blastocyst transfer compared to transfer of cleavage stage embryos (Blake et al., 2007).

Currently, increasing number of legislation acts on MAR in developed countries impose mandatory single embryo transfer under certain circumstances (mostly in younger patients), in order to decrease the incidence of multiple gestations. Thus, the question remains; how to select the best embryos in order to achieve the highest success and at the same time lower the incidence of multiple gestations? With regards to these facts, additional studies should settle the issue in what scenario can the number of oocytes be a factor in decision for SET or multiple embryo transfer and whether embryos should be transferred at cleavage or blastocyst stage.

## **4. Influence of number of retrieved oocytes on embryo transfer success**

A retrospective study of retrieved oocyte number on MAR outcome was performed at Department of Reproductive Medicine and Gynaecologic Endocrinology, University Medical Centre in Maribor. Totally, 6989 consecutive in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles resulting in follicle aspiration and oocyte pick-up were included. Ovarian stimulation protocols with combination of GnRH agonist/GnRH antagonist and recombinant FSH (Gonal-f®, Serono International SA, Geneva, Switzerland)/HMG (Menopur®, Ferring Pharmaceuticals Inc., Saint-Prex, Switzerland) were used and were previously described in detail (Vlaisavljević et al., 2000). Embryo quality was assessed by an experienced embryologist at day two and blastocysts were graded according to our established grading system at day five after oocyte pick-up (Kovačič et al., 2004). Embryo transfer was carried out three or five days after oocyte pick-up. Day five blastocyst transfer was performed if more than four fertilised oocytes were obtained and if more than three optimal embryos were available on day three according to our standard policies (Kovačič et al., 2002). After consultation with the patients, time of embryo transfer could be adjusted to

Does the Number of Retrieved Oocytes Influence

No of previous MAR

No. of oocytes

Fertilised oocytes

Percentage of day 5

Cryopreserved

Cycles with blastocyst

Pregnancy Rate After Day 3 and Day 5 Embryo Transfer? 43

No. of oocytes **1-5 6-10 11-15 16-20 >20 TOTAL**  No. of cycles 2396 2405 1269<PHONE_NUMBER> Age (SD) 36.1 (4.6)a 33.8 (4.6)b 32.6 (4.2)c 32.2 (4.4)c 32.0 (4.2)c 34.2 (4.7)

attempts (SD) 2.0 (2.4)a 1.6 (2.1)b 1.5 (1.9)bc 1.4 (1.9)c 1.3 (1.6)c 1.7 (2.2)

collected 7765 18823 16061 9571 9573 61793

(2PN) 4910 11552 9668 5686 5510 37326 Day 2 embryos 4837 11352 9454 5565 5381 36589 ET(s) 2027 2241 1213<PHONE_NUMBER>

ETs 14.0%a 69.4%b 86.7%c 92.6%d 94.3%d 58.2% Cancelled ET 15.4%a 6.8%b 4.4%c 5.7%bc 11.5%ab 9.5% Transferred embryos 3502 4040 2123<PHONE_NUMBER>2

embryos 295 2212 2544 1982 2150 9183 Clinical Pregnancies 532 954 638<PHONE_NUMBER> Avg. Fertilisation rate 75.6%a 77.2%ab 77.6%ab 79.3%ab 83.8%b 77.3% Pregnancy rate/cycle 22.2%a 39.7%b 50.3%c 52.6%c 49.1%c 37.1% Pregnancy rate/ET 26.3%a 42.6%b 52.6%c 55.8%c 55.4%c 41.0%

cryopreservation 8.3%a 38.9%b 58.3%c 73.0%d 83.5%e 37.0%

**4.3 Clinical pregnancy & delivery rates in relation to the number of retrieved oocytes**  Clinical pregnancy and delivery rates were calculated per each number of collected oocytes. Clinical pregnancy rate rises constantly to peak at 11-15 oocytes and from then on remains constant until it declines slightly at high responders with more than 20 oocytes (Table 1 & Figure 2). Nonetheless, there are no statistically significant changes in pregnancy rates in groups of patients with more then 11-15 harvested oocytes. However, the risks related to ovarian stimulation increase with rising oocyte count, which is partially reflected in higher cycle cancellation rates, especially in patients with more than 20 harvested oocytes (Table 1). Published data about the number of oocytes influencing embryo transfer outcome are not consistent. Whilst some scientists claim that oocyte number plays no role in achievement of pregnancy after embryo transfer (Letterie et al., 2005; Yoldemir et al., 2010), others report of increasing pregnancy rates with increasing number of oocytes. The optimal numbers reported in these studies are usually in the range of 5 to 15 oocytes (van Gast et al., 2006; Meniru & Craft, 1997; Timeva et al., 2006). Most of these studies are however single-centre analyses performed on a fairly low number of patients. Recently though, Sunkara et al performed a large scale review of UK national IVF data and concluded that 15 is the optimal number of oocytes to be collected at retrieval (Sunkara et al., 2011). Results of our study that

abcde Within each category, numbers with different letter superscripts are significantly different from each other, numbers with the same letter superscript are not significantly different (*p*<0.05). Table 1. Outcome of MAR treatment according to the number of retrieved oocytes

day 3 or day 5 according to doctor-patient agreement. Clinical pregnancy was defined as the presence of fetal heartbeat on ultrasound examination at six weeks of gestation. Frozenthawed embryo transfer cycles and cycles with preimplantation genetic diagnostics (PGD) or in vitro maturated (IVM) oocytes were excluded from the study.

Patients were stratified according to the number of oocytes collected at retrieval. Categorical variables were tested using Chi-Square test. Numerical values were tested bivariately using Student's *t*-test or Mann-Whitney *U* test. The analysis of variance test (ANOVA) with Tukey HSD or Games-Howell post-hoc test was used for analysing differences in continuous variables between groups stratified by the number of retrieved oocytes. To further explore the association between the number of oocytes and clinical pregnancy rate, a logistic regression model was constructed. The model was adjusted for confounding variables that affected pregnancy rate in univariate analysis (the history of previous MAR attempts, age and dose of gonadotrophins). Data were analysed using SPSS 17.0 (SPSS Inc., Chicago IL) statistical software package.

#### **4.1 Distribution of retrieved oocytes per cycle**

In 6989 studied cycles, 61793 oocytes were collected at retrieval. The median number of retrieved oocytes was 8 [interquartile range (IQR) 4-12] and the median number of embryos created was 4 [IQR 2-7]. Figure 1 represents the distribution of collected oocytes in cycles resulting in oocyte pick-up.

Fig. 1. Distribution of retrieved oocytes.

#### **4.2 Outcome of MAR cycles**

Cycles were stratified according to the number of retrieved oocytes. Overall clinical pregnancy rate per cycle was 37.1%, calculated pregnancy rate per embryo transfer was 41.0%. Delivery rates per cycle and per embryo transfer were calculated to be 29.8% and 32.9%, respectively. Clinical outcomes are summarized in Table 1.

day 3 or day 5 according to doctor-patient agreement. Clinical pregnancy was defined as the presence of fetal heartbeat on ultrasound examination at six weeks of gestation. Frozenthawed embryo transfer cycles and cycles with preimplantation genetic diagnostics (PGD) or

Patients were stratified according to the number of oocytes collected at retrieval. Categorical variables were tested using Chi-Square test. Numerical values were tested bivariately using Student's *t*-test or Mann-Whitney *U* test. The analysis of variance test (ANOVA) with Tukey HSD or Games-Howell post-hoc test was used for analysing differences in continuous variables between groups stratified by the number of retrieved oocytes. To further explore the association between the number of oocytes and clinical pregnancy rate, a logistic regression model was constructed. The model was adjusted for confounding variables that affected pregnancy rate in univariate analysis (the history of previous MAR attempts, age and dose of gonadotrophins). Data were analysed using SPSS 17.0 (SPSS Inc., Chicago IL)

In 6989 studied cycles, 61793 oocytes were collected at retrieval. The median number of retrieved oocytes was 8 [interquartile range (IQR) 4-12] and the median number of embryos created was 4 [IQR 2-7]. Figure 1 represents the distribution of collected oocytes in cycles

Cycles were stratified according to the number of retrieved oocytes. Overall clinical pregnancy rate per cycle was 37.1%, calculated pregnancy rate per embryo transfer was 41.0%. Delivery rates per cycle and per embryo transfer were calculated to be 29.8% and

32.9%, respectively. Clinical outcomes are summarized in Table 1.

in vitro maturated (IVM) oocytes were excluded from the study.

statistical software package.

resulting in oocyte pick-up.

Fig. 1. Distribution of retrieved oocytes.

**4.2 Outcome of MAR cycles**

**4.1 Distribution of retrieved oocytes per cycle**

abcde Within each category, numbers with different letter superscripts are significantly different from each other, numbers with the same letter superscript are not significantly different (*p*<0.05).

Table 1. Outcome of MAR treatment according to the number of retrieved oocytes

#### **4.3 Clinical pregnancy & delivery rates in relation to the number of retrieved oocytes**

Clinical pregnancy and delivery rates were calculated per each number of collected oocytes. Clinical pregnancy rate rises constantly to peak at 11-15 oocytes and from then on remains constant until it declines slightly at high responders with more than 20 oocytes (Table 1 & Figure 2). Nonetheless, there are no statistically significant changes in pregnancy rates in groups of patients with more then 11-15 harvested oocytes. However, the risks related to ovarian stimulation increase with rising oocyte count, which is partially reflected in higher cycle cancellation rates, especially in patients with more than 20 harvested oocytes (Table 1). Published data about the number of oocytes influencing embryo transfer outcome are not consistent. Whilst some scientists claim that oocyte number plays no role in achievement of pregnancy after embryo transfer (Letterie et al., 2005; Yoldemir et al., 2010), others report of increasing pregnancy rates with increasing number of oocytes. The optimal numbers reported in these studies are usually in the range of 5 to 15 oocytes (van Gast et al., 2006; Meniru & Craft, 1997; Timeva et al., 2006). Most of these studies are however single-centre analyses performed on a fairly low number of patients. Recently though, Sunkara et al performed a large scale review of UK national IVF data and concluded that 15 is the optimal number of oocytes to be collected at retrieval (Sunkara et al., 2011). Results of our study that

Does the Number of Retrieved Oocytes Influence

of mild stimulation protocols in routine MAR treatment.

No of previous MAR

Cycles with blastocyst

**4.4 Effect of age**

Pregnancy Rate After Day 3 and Day 5 Embryo Transfer? 45

performed meta-analysis included only three studies featuring low number of participants (Verberg et al., 2009). All of the studies included report of relatively low pregnancy rates, 15- 21%. What is more, the studies did not account for possible added benefits of embryo cryopreservation, which could shift the scale to the side of classic ovarian stimulation even further if adding freeze-thaw cycles to the analysis. In theory, mild stimulation seems a feasible choice that could be especially indicated in poor responding patients. Because of lower gonadotrophin dose, higher quality of oocytes and endometrium could be expected. But since there is very limited data available, current reports do not provide enough supporting evidence and in the end, additional studies are needed in order to define the role

Number of oocytes **1 2-3 4-5 TOTAL** No. of cycles<PHONE_NUMBER> 2396

No of oocytes collected 305 2467 4993 7765 Fertilised oocytes (2PN) 212 1574 3124 4910 Day 2 embryos 205 1532 3100 4837 ET(s)<PHONE_NUMBER> 2027 Percentage of day 5 ETs 0.5% 3.0% 25.4% 14.0%

Transferred embryos 186 1368 1948 3502 Cryopreserved embryos 0 41 254 295 Clinical Pregnancies 16 184 332 532

other, numbers with the same letter superscript are not significantly different.

Age (SD) 37.9 (4.2)a 36.7 (4.4)b 35.2 (4.6)c 36.1 (4.6) *p*<0.001

attempts (SD) 2.0 (2.4)a 2.1 (2.7)a 1.9 (2.2)a 2.0 (2.4) *<sup>p</sup>*>0.05 Gonadotrophin used (IE) 2979a 2789b 2518c 2692 *p*<0.001

Cancelled ET 39.34%a 15.77%b 8.48%c 15.40% *p*<0.001

Avg. Fertilisation rate 78.2%a 75.0%a 75.8%a 75.6% *p*>0.05 Pregnancy rate/cycle 5.30%a 18.70%b 30.00%c 22.20% *p*<0.001 Pregnancy rate/ET 8.70%a 22.20%b 32.70%c 26.30% *p*<0.001

cryopreservation 0%a 3.30%b 15.10%c 8.30% *p*<0.001

abc Within each category, numbers with different letter superscripts are significantly different from each

Besides the number of oocytes, age was found to be one of the most important factors in predicting the success of the started cycle in our data. In further analysis, cycles were stratified according to the age of female patients. Advancing age significantly adversely affects the outcome of MAR cycles independently of oocyte count. Average clinical pregnancy rate was the highest in the youngest group of patients (20-34 years) at 45.6% and decreased to 16.8% in women over 40 years of age. On the other hand, mean oocyte count was also significantly lower in older women (20-34 years; 10.4 oocytes, 35-37 years; 8.5 oocytes, 38-39 years; 6.9

Table 2. Cycle characteristics in "poor" and "low" responders (<5 oocytes collected)

oocytes, 40-44 years; 5.7 oocytes, *p*<0.001). The results are illustrated in Figure 3.

included nearly 7000 cycles suggest that the optimal number of oocytes to aim for at ovarian stimulation in order to achieve clinical pregnancy should be between 11 and 15.

Fig. 2. Clinical pregnancy and delivery rates in relation to the number of retrieved oocytes

## **4.3.1 Low number of oocytes**

As noted above, pregnancy and delivery rates are significantly lower in cycles where less than 5 oocytes are retrieved at pickup. Accordingly, these cycles were analysed more thoroughly. Significant changes can be observed in pregnancy rates among each of the groups (Table 2). These differences remain significant even after controlling for the age factor and history of previous MAR attempts in multivariate analysis.

It has been demonstrated that low oocyte numbers after ovarian stimulation are related to ovarian ageing and the depletion of primordial follicle pool (Tarlatzis et al., 2003). Ovarian stimulation for women with low ovarian reserve has remained one of the most frustrating aspects of IVF (Revelli et al., 2011). However, direct correlation with pregnancy rate has not been thoroughly investigated.

Low oocyte number coupled with high gonadotrophin dose in conventional stimulation methods can imply lower oocyte quality. Furthermore, endometrial quality can also be hampered in high-dose stimulating protocols (Gougeon, 1996; Pal et al., 2008). On the other hand, higher number of oocytes simply allows for better selection of quality embryos from a larger cohort of available embryos (Devreker, 1999; Pal et al., 2008).

On the opposite, it has been theorised that milder forms of ovarian stimulation allow for higher quality oocytes and embryos with lower incidence of chromosome aneuploidies that can result in comparable pregnancy rates in spite of the lower number of harvested oocytes (Verberg et al., 2009). However, studies on this subject are relatively sparse and a recently

included nearly 7000 cycles suggest that the optimal number of oocytes to aim for at ovarian

Fig. 2. Clinical pregnancy and delivery rates in relation to the number of retrieved oocytes

As noted above, pregnancy and delivery rates are significantly lower in cycles where less than 5 oocytes are retrieved at pickup. Accordingly, these cycles were analysed more thoroughly. Significant changes can be observed in pregnancy rates among each of the groups (Table 2). These differences remain significant even after controlling for the age

It has been demonstrated that low oocyte numbers after ovarian stimulation are related to ovarian ageing and the depletion of primordial follicle pool (Tarlatzis et al., 2003). Ovarian stimulation for women with low ovarian reserve has remained one of the most frustrating aspects of IVF (Revelli et al., 2011). However, direct correlation with pregnancy rate has not

Low oocyte number coupled with high gonadotrophin dose in conventional stimulation methods can imply lower oocyte quality. Furthermore, endometrial quality can also be hampered in high-dose stimulating protocols (Gougeon, 1996; Pal et al., 2008). On the other hand, higher number of oocytes simply allows for better selection of quality embryos from a

On the opposite, it has been theorised that milder forms of ovarian stimulation allow for higher quality oocytes and embryos with lower incidence of chromosome aneuploidies that can result in comparable pregnancy rates in spite of the lower number of harvested oocytes (Verberg et al., 2009). However, studies on this subject are relatively sparse and a recently

factor and history of previous MAR attempts in multivariate analysis.

larger cohort of available embryos (Devreker, 1999; Pal et al., 2008).

**4.3.1 Low number of oocytes**

been thoroughly investigated.

stimulation in order to achieve clinical pregnancy should be between 11 and 15.

performed meta-analysis included only three studies featuring low number of participants (Verberg et al., 2009). All of the studies included report of relatively low pregnancy rates, 15- 21%. What is more, the studies did not account for possible added benefits of embryo cryopreservation, which could shift the scale to the side of classic ovarian stimulation even further if adding freeze-thaw cycles to the analysis. In theory, mild stimulation seems a feasible choice that could be especially indicated in poor responding patients. Because of lower gonadotrophin dose, higher quality of oocytes and endometrium could be expected. But since there is very limited data available, current reports do not provide enough supporting evidence and in the end, additional studies are needed in order to define the role of mild stimulation protocols in routine MAR treatment.

abc Within each category, numbers with different letter superscripts are significantly different from each other, numbers with the same letter superscript are not significantly different.

Table 2. Cycle characteristics in "poor" and "low" responders (<5 oocytes collected)

#### **4.4 Effect of age**

Besides the number of oocytes, age was found to be one of the most important factors in predicting the success of the started cycle in our data. In further analysis, cycles were stratified according to the age of female patients. Advancing age significantly adversely affects the outcome of MAR cycles independently of oocyte count. Average clinical pregnancy rate was the highest in the youngest group of patients (20-34 years) at 45.6% and decreased to 16.8% in women over 40 years of age. On the other hand, mean oocyte count was also significantly lower in older women (20-34 years; 10.4 oocytes, 35-37 years; 8.5 oocytes, 38-39 years; 6.9 oocytes, 40-44 years; 5.7 oocytes, *p*<0.001). The results are illustrated in Figure 3.

Does the Number of Retrieved Oocytes Influence

the absence of ovarian hyperstimulation syndrome risk.

Pregnancy Rate After Day 3 and Day 5 Embryo Transfer? 47

results, recently performed studies argue that the simple aim to harvest as many oocytes as possible is simply not justified (van der Gast et al., 2006; Sunkara et al., 2011). Our analysis has some limitations due to the fact that incidence of OHSS could not be analysed and cycles with cryopreservation of the whole embryo cohort due to high OHSS risk could not be excluded from the analysis. It could be reasoned that lower pregnancy rates and high embryo transfer cancellation can be attributed to ovarian hyperstimulation syndrome risk. But considering data in the literature, approximately 15% incidence of OHSS can be predicted at 20 collected oocytes and the risk increases with rising oocyte count (Reljič et al., 1999; Verwoerd et al., 2008). In addition, there is also evidence that high estradiol levels may negatively impact developmental potential of the embryos (Ertzeid & Storeng, 2001) and interfere with endometrial receptivity in the early luteal phase (Devroey et al., 2004; Horcajadas et al., 2008). Thus, pregnancy rates can be hampered in high responders even in

Fig. 4. Association of the number of retrieved oocytes to embryo cryopreservation, successful blastocyst culture and proportion of good quality embryos on day two.

Nonetheless, an increase in proportion of cycles with embryos available for freezing can be observed up until ~20 oocytes (Table 1 & Figure 4). The relationship was evident also in multivariate analysis after controlling for possible confounding variables (age, history of previous MAR attempts and dosage of gonadotrophins). Somewhat lower pregnancy rates in these patients could theoretically be recovered in subsequent cryo-thawed cycles. It should be noted though, that this group of patients included also couples with cancelled embryo transfer and cryopreservation of the whole embryo cohort due to high OHSS risk. Due to our study design, linkage between fresh and cryo-thawed cycles was not possible and the analysis of cumulative pregnancy rates could not be performed. Our results are comparable with findings of the research by Hamoda et al in which there was no increment in cycles with available oocytes for freezing beyond 18 harvested oocytes (Figure 4).

Fig. 3. Association between oocyte number and delivery rate stratified by the female patient age.

With ovarian ageing, the depletion of ovarian reserve usually involves adjusting stimulation protocols with higher doses of gonadotrophins that result in negative effects as discussed in previous topic. Although our study did not consider antral follicle count (AFC) and anti-Müllerian hormone (AMH) as a measure of ovarian reserve, it can be clearly seen from our results that age is independent predictor of lower oocyte count at retrieval. But even in the case of normal response to gonadotrophin treatment in older women, pregnancy rates are lower compared to younger patients. This could be related to the high proportion of embryo aneuploidies in these patients. This problem grows quickly after 40 years of age and after the age of 45 the birth of a healthy baby after MAR is very rare in spite of normal ovarian reserve tests (Forman et al., 2011).

#### **4.5 Embryo cryopreservation**

Although the birth of a live baby preceded by the successful achievement of clinical pregnancy is the single most important outcome of MAR treatment, several other surrogate indicators were evaluated in order to assess the quality of MAR cycles.

Embryo transfer was performed in 91.5% of cycles after oocyte pick-up. In approximately 9.5% cycles transfer had to be cancelled. The rate of transfer cancellation is the highest in "poor" responding patients and approximately 40% of transfers were cancelled when only 1 oocyte was retrieved (Table 1 & Table 2). This can be contributed to the fact that there are fewer embryos available for transfer and also to the lower quality of oocytes available for treatment (Tarlatzis et al., 2003). On the other hand, cycle cancellation again increases significantly in high responding patients, with more than 20 oocytes collected at retrieval. Together with the higher rate of cycle cancellation, a drop in pregnancy and delivery rates is also observed beyond 20 harvested oocytes (Table 1 & Figure 2). In concordance with these

Fig. 3. Association between oocyte number and delivery rate stratified by the female

With ovarian ageing, the depletion of ovarian reserve usually involves adjusting stimulation protocols with higher doses of gonadotrophins that result in negative effects as discussed in previous topic. Although our study did not consider antral follicle count (AFC) and anti-Müllerian hormone (AMH) as a measure of ovarian reserve, it can be clearly seen from our results that age is independent predictor of lower oocyte count at retrieval. But even in the case of normal response to gonadotrophin treatment in older women, pregnancy rates are lower compared to younger patients. This could be related to the high proportion of embryo aneuploidies in these patients. This problem grows quickly after 40 years of age and after the age of 45 the birth of a healthy baby after MAR is very rare in spite of normal ovarian

Although the birth of a live baby preceded by the successful achievement of clinical pregnancy is the single most important outcome of MAR treatment, several other surrogate

Embryo transfer was performed in 91.5% of cycles after oocyte pick-up. In approximately 9.5% cycles transfer had to be cancelled. The rate of transfer cancellation is the highest in "poor" responding patients and approximately 40% of transfers were cancelled when only 1 oocyte was retrieved (Table 1 & Table 2). This can be contributed to the fact that there are fewer embryos available for transfer and also to the lower quality of oocytes available for treatment (Tarlatzis et al., 2003). On the other hand, cycle cancellation again increases significantly in high responding patients, with more than 20 oocytes collected at retrieval. Together with the higher rate of cycle cancellation, a drop in pregnancy and delivery rates is also observed beyond 20 harvested oocytes (Table 1 & Figure 2). In concordance with these

indicators were evaluated in order to assess the quality of MAR cycles.

patient age.

reserve tests (Forman et al., 2011).

**4.5 Embryo cryopreservation**

results, recently performed studies argue that the simple aim to harvest as many oocytes as possible is simply not justified (van der Gast et al., 2006; Sunkara et al., 2011). Our analysis has some limitations due to the fact that incidence of OHSS could not be analysed and cycles with cryopreservation of the whole embryo cohort due to high OHSS risk could not be excluded from the analysis. It could be reasoned that lower pregnancy rates and high embryo transfer cancellation can be attributed to ovarian hyperstimulation syndrome risk. But considering data in the literature, approximately 15% incidence of OHSS can be predicted at 20 collected oocytes and the risk increases with rising oocyte count (Reljič et al., 1999; Verwoerd et al., 2008). In addition, there is also evidence that high estradiol levels may negatively impact developmental potential of the embryos (Ertzeid & Storeng, 2001) and interfere with endometrial receptivity in the early luteal phase (Devroey et al., 2004; Horcajadas et al., 2008). Thus, pregnancy rates can be hampered in high responders even in the absence of ovarian hyperstimulation syndrome risk.

Fig. 4. Association of the number of retrieved oocytes to embryo cryopreservation, successful blastocyst culture and proportion of good quality embryos on day two.

Nonetheless, an increase in proportion of cycles with embryos available for freezing can be observed up until ~20 oocytes (Table 1 & Figure 4). The relationship was evident also in multivariate analysis after controlling for possible confounding variables (age, history of previous MAR attempts and dosage of gonadotrophins). Somewhat lower pregnancy rates in these patients could theoretically be recovered in subsequent cryo-thawed cycles. It should be noted though, that this group of patients included also couples with cancelled embryo transfer and cryopreservation of the whole embryo cohort due to high OHSS risk. Due to our study design, linkage between fresh and cryo-thawed cycles was not possible and the analysis of cumulative pregnancy rates could not be performed. Our results are comparable with findings of the research by Hamoda et al in which there was no increment in cycles with available oocytes for freezing beyond 18 harvested oocytes (Figure 4).

Does the Number of Retrieved Oocytes Influence

**(4.3)**

**11.8 (5.6)**

pregnancy/ET **43.1%** (625)

Twins **24.5%** (8)

Triplets **0.4%** 0 (5)

 »SET«: single cleavage stage embryo transfer »DET«: double cleavage stage embryo transfer »TET«: triple cleavage stage embryo transfer

even in the case of dealing with older patients.

Table 3. Embryo cultivation strategies and clinical outcome

Legend: »SBT«: single blastocyst transfer »DBT«: double blastocyst transfer

31.8 (4.2)a

12.5 (6.5)a

43.1%a

1.7%

blastocyst transfer (38.1%).

Age (SD) **32.8**

Avg. no of oocytes (SD)

Clinical

Ongoing

Implantation

% transfers with optimal quality blastocyst

Pregnancy Rate After Day 3 and Day 5 Embryo Transfer? 49

Additionally, it can clearly be seen that double blastocyst transfer does not improve pregnancy rates compared to transfer of a single blastocyst. This can be observed even when analysing only cycles with at least one optimal blastocyst transferred. On the other hand, the incidence of twin deliveries rises dramatically with double embryo, especially double

No. of cycles **3572** 1452 2120 **2647** 798 1377 472 **6219**

pregnancy/ET **52.7%** 52.2%a 53.1%a **25.3%** 15.2%b 30.5%c 27.3%c **41.1%**

rate **43.9%** 53.1%a 37.6%b **16.6%** 15.1%cd 19.2%c 11.3%d **31.2%**

abcde Within each row, numbers with different letter superscripts are significantly different from each

The detailed investigation of the incidence of multiple gestations reveals that the incidence of higher order multiple pregnancies was low, as only 6 triplet pregnancies were observed during the ten-year study period. Conversely, birth of twins was recorded in 22.8% of deliveries and as mentioned before, this was especially high in the transfer of two blastocysts. In our publications, it was demonstrated that the age of the female patient and additional spare blastocysts available for cryopreservation are an important risk factor for multiple gestation after blastocyst transfer (Vlaisavljević et al., 2004). Our current data has shown, that even in the group of patients older than 40 years, after double blastocyst transfer, there were 27.7% twin gestations. This exemplifies the fact that the only option to minimize the risk of multiple gestations is strict implementation of single embryo transfer,

43.2%a **18.6%** (92)

38.1% **17.6%** (1)

**35.8 (4.6)**

**5.2 (3.7)**

**53.2%** 60.6% 48.2% . . . **.**

33.4 (4.3)b

11.3 (5.1)b

(912)

(303)

other, numbers with the same letter superscript are not significantly different (*p*<0.05)

**Day 5 SBT DBT Day 3 SET DET TET TOTAL**

35.7 (4.7)c

3.5 (2.8)c

11.6%b

1.4%

0.8% **0.3%** 0 (1)

35.3 (4.5)c

5.6 (3.6)d

(313) 22.8%c

(49) 20.1%

37.5 (4.3)d

6.9 (4.0)e

(84) 17.9%c

(19) 25.3%

0.4% 0 **(6)**

**8.8 (6.1)**

**(1663) 32.9%**

**(380) 22.8%**

**0.4%**

Furthermore, the storage of high numbers of cryopreserved embryos can also lead to logistic, administrative and ethical problems. Even so, in a unit with a successful cryopreservation programme, especially with the advances of embryo vitrification, cumulative pregnancy rates can be substantially improved (Kolibianakis et al., 2009).

Additionally, blastocyst transfer was performed significantly more frequently with rising number of harvested oocytes. Because day 5 blastocyst transfer allows for enhanced embryo selection and can optimize the chance of embryo transfer success (Blake et al., 2007; Kovačič et al, 2002; Papanikolaou et al., 2008), further analysis was aimed toward the comparison of different embryo cultivation and transfer policies.

#### **4.6 Embryo cultivation protocol**

Couples were stratified according to the stage at which embryos were replaced in the uterus. At blastocyst stage, single embryo or two embryos were transferred, at cleavage stage one to three embryos were transferred depending on the age, history of the patients and embryo quality. Results of embryo transfer after different protocols are illustrated in Table 3.

In the beginnings of MAR, cleavage stage embryo transfer was traditionally performed. Physiology and energy metabolism of early embryos were not well understood and thus media could not be used to culture embryos beyond the four-cell stage. However, with scientific advances, new improved, sequential blastocyst media have enabled longer in vitro cultivation of embryos (Menezo et al., 1998). There are two central reasons why this should theoretically improve embryo transfer results. First of all, it is considered that blastocyst transfer mimics natural conception physiology as the embryo travels through Fallopian tubes and reaches the uterine cavity no sooner than the fourth day after conception. The uterus provides different nutritional conditions for the embryo and this may cause homeostatic stress and reduce embryo implantation rates after cleavage stage transfer (Blake et al., 2007). The second reason lies in the before mentioned chance of better morphologic selection of the embryos at blastocyst stage. However, in our previous studies, it was demonstrated that in the case of low number of embryos this advantages did not help to improve success rates of embryo transfer. Extended blastocyst culture did not prove to be of any value in improving pregnancy rates in the scenario when fewer than three embryos were available at day two after oocyte pickup. On the other hand, although transfer cancellation was significantly higher when waiting until day five for transfer, pregnancy and delivery rates per started cycle were comparable (Kovačič et al., 2002; Vlaisavljević et al., 2001). According to the present study, clinical and ongoing pregnancy rates were significantly higher in the case of blastocyst transfer compared to day three cleavage stage embryo transfer (52.7% vs. 25.3% and 43.1% vs. 18.6% respectively). Even in the multivariable logistic regression model, adjusting for the age of the patients and the number of harvested oocytes, these differences remained statistically significant. These results should however be interpreted in the light of the fact that these groups were composed of patients with uneven characteristics. Generally, patients with less than five harvested oocytes and fewer than three embryos on day two that have effectively worse prognosis underwent cleavage stage transfer on day three. The number of harvested oocytes was significantly lower in day 3 group transfer as compared to day 5. In spite of this, considering data in the literature (Blake et al., 2007; Papanikolaou et al., 2008), our data confirm that blastocyst transfer provides a higher chance of embryo implantation and subsequent clinical pregnancy.

Furthermore, the storage of high numbers of cryopreserved embryos can also lead to logistic, administrative and ethical problems. Even so, in a unit with a successful cryopreservation programme, especially with the advances of embryo vitrification,

Additionally, blastocyst transfer was performed significantly more frequently with rising number of harvested oocytes. Because day 5 blastocyst transfer allows for enhanced embryo selection and can optimize the chance of embryo transfer success (Blake et al., 2007; Kovačič et al, 2002; Papanikolaou et al., 2008), further analysis was aimed toward the comparison of

Couples were stratified according to the stage at which embryos were replaced in the uterus. At blastocyst stage, single embryo or two embryos were transferred, at cleavage stage one to three embryos were transferred depending on the age, history of the patients and embryo

In the beginnings of MAR, cleavage stage embryo transfer was traditionally performed. Physiology and energy metabolism of early embryos were not well understood and thus media could not be used to culture embryos beyond the four-cell stage. However, with scientific advances, new improved, sequential blastocyst media have enabled longer in vitro cultivation of embryos (Menezo et al., 1998). There are two central reasons why this should theoretically improve embryo transfer results. First of all, it is considered that blastocyst transfer mimics natural conception physiology as the embryo travels through Fallopian tubes and reaches the uterine cavity no sooner than the fourth day after conception. The uterus provides different nutritional conditions for the embryo and this may cause homeostatic stress and reduce embryo implantation rates after cleavage stage transfer (Blake et al., 2007). The second reason lies in the before mentioned chance of better morphologic selection of the embryos at blastocyst stage. However, in our previous studies, it was demonstrated that in the case of low number of embryos this advantages did not help to improve success rates of embryo transfer. Extended blastocyst culture did not prove to be of any value in improving pregnancy rates in the scenario when fewer than three embryos were available at day two after oocyte pickup. On the other hand, although transfer cancellation was significantly higher when waiting until day five for transfer, pregnancy and delivery rates per started cycle were comparable (Kovačič et al., 2002; Vlaisavljević et al., 2001). According to the present study, clinical and ongoing pregnancy rates were significantly higher in the case of blastocyst transfer compared to day three cleavage stage embryo transfer (52.7% vs. 25.3% and 43.1% vs. 18.6% respectively). Even in the multivariable logistic regression model, adjusting for the age of the patients and the number of harvested oocytes, these differences remained statistically significant. These results should however be interpreted in the light of the fact that these groups were composed of patients with uneven characteristics. Generally, patients with less than five harvested oocytes and fewer than three embryos on day two that have effectively worse prognosis underwent cleavage stage transfer on day three. The number of harvested oocytes was significantly lower in day 3 group transfer as compared to day 5. In spite of this, considering data in the literature (Blake et al., 2007; Papanikolaou et al., 2008), our data confirm that blastocyst transfer provides a higher chance of embryo implantation and

quality. Results of embryo transfer after different protocols are illustrated in Table 3.

cumulative pregnancy rates can be substantially improved (Kolibianakis et al., 2009).

different embryo cultivation and transfer policies.

**4.6 Embryo cultivation protocol**

subsequent clinical pregnancy.

Additionally, it can clearly be seen that double blastocyst transfer does not improve pregnancy rates compared to transfer of a single blastocyst. This can be observed even when analysing only cycles with at least one optimal blastocyst transferred. On the other hand, the incidence of twin deliveries rises dramatically with double embryo, especially double blastocyst transfer (38.1%).

abcde Within each row, numbers with different letter superscripts are significantly different from each other, numbers with the same letter superscript are not significantly different (*p*<0.05)

Legend: »SBT«: single blastocyst transfer »DBT«: double blastocyst transfer »SET«: single cleavage stage embryo transfer

»DET«: double cleavage stage embryo transfer

»TET«: triple cleavage stage embryo transfer

Table 3. Embryo cultivation strategies and clinical outcome

The detailed investigation of the incidence of multiple gestations reveals that the incidence of higher order multiple pregnancies was low, as only 6 triplet pregnancies were observed during the ten-year study period. Conversely, birth of twins was recorded in 22.8% of deliveries and as mentioned before, this was especially high in the transfer of two blastocysts. In our publications, it was demonstrated that the age of the female patient and additional spare blastocysts available for cryopreservation are an important risk factor for multiple gestation after blastocyst transfer (Vlaisavljević et al., 2004). Our current data has shown, that even in the group of patients older than 40 years, after double blastocyst transfer, there were 27.7% twin gestations. This exemplifies the fact that the only option to minimize the risk of multiple gestations is strict implementation of single embryo transfer, even in the case of dealing with older patients.

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number, and success in oocyte donor cycles. *Journal of Assisted Reproduction and*

ovarian stimulation for in vitro fertilization. *Endocrine Reviews*, Vol.27, No.2, pp.

tosequential defined media for transfer at the blastocyst stage. *Human Reproduction*,

of superovulation strategies for in-vitro fertilization treatment. *Human Reproduction*,

for ovarian stimulation adversely influences clinical pregnancy and live birth after

Devroey P. (2008). Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. *Human*

## **5. Conclusion**

Introduction of exogenous gonadotrophins to ovarian stimulation represents one of the most important events in MAR that substantially improved the results of infertility treatment. The first successful pregnancies achieved with MAR were the result of a natural cycle IVF. Since then, ovarian stimulation protocols using GnRH agonists or antagonists in the combination with recombinant or highly purified gonadotrophins have become an everyday routine in MAR practice. Our results show that current protocols of ovarian stimulation should be used in a way to avoid excessive follicular development and to achieve moderate stimulation of the ovaries. This approach provides superior results compared to either aggressive or mild stimulation protocols. According to our study, oocyte pick-up resulting in eleven to fifteen harvested oocytes presents the optimal outcome of ovarian stimulation. Day five blastocyst transfer enables for higher pregnancy rates compared to transfer of cleavage stage embryos, especially when high number of developed embryos is available on day three after oocyte pick-up. Extended blastocyst culture also allows for transfer of reduced number of embryos without decreasing the overall pregnancy rate. However, only strict implementation of single embryo transfer can lead to a decrease in the incidence of multiple gestations. These steps, coupled with successful laboratory embryo cryopreservation programme, present as an optimal choice for MAR.

#### **6. References**

Introduction of exogenous gonadotrophins to ovarian stimulation represents one of the most important events in MAR that substantially improved the results of infertility treatment. The first successful pregnancies achieved with MAR were the result of a natural cycle IVF. Since then, ovarian stimulation protocols using GnRH agonists or antagonists in the combination with recombinant or highly purified gonadotrophins have become an everyday routine in MAR practice. Our results show that current protocols of ovarian stimulation should be used in a way to avoid excessive follicular development and to achieve moderate stimulation of the ovaries. This approach provides superior results compared to either aggressive or mild stimulation protocols. According to our study, oocyte pick-up resulting in eleven to fifteen harvested oocytes presents the optimal outcome of ovarian stimulation. Day five blastocyst transfer enables for higher pregnancy rates compared to transfer of cleavage stage embryos, especially when high number of developed embryos is available on day three after oocyte pick-up. Extended blastocyst culture also allows for transfer of reduced number of embryos without decreasing the overall pregnancy rate. However, only strict implementation of single embryo transfer can lead to a decrease in the incidence of multiple gestations. These steps, coupled with successful laboratory embryo

Asch, RH. Li, HP. Balmaceda, JP. Weckstein LN & Stone, SC. (1991). Severe ovarian

Blake, D. Farquhar, C. Johnson, N. & Proctor, M. (2007). Cleavage stage versus blastocyst stage

*Reviews,* No.4, Art. No.: CD002118. DOI: 10.1002/14651858.CD002118.pub3. Brinsden, PR. Wada, I. Tan, SL. Balen, A & Jacobs, HS. (1995). Diagnosis, prevention and

Devreker, F. Pogonici, E. De Maertelaer, V., Revelard, P., Van den Bergh, M. & Englert Y.

Devroey, P. Bourgain, C. Macklon, NS. & Fauser, BC. (2004). Reproductive biology and IVF:

Edwards, RG. Steptoe, PC. & Purdy, JM. (1980). Establishing full-term human pregnancies

Edwards, RG. (2007). IVF, IVM, natural cycle IVF, minimal stimulation IVF – tome for a

Ertzeid, G & Storeng, R. (2001). The impact of ovarian stimulation on implantation and fetal

Fauser, BC. & van Heusden, AM. (1997). Manipulation of human ovarian function:

rethink. *Reproductive Biomedicine Online,* Vol.15, No.1, pp. 106-19

development in mice. *Human Reproduction*, Vol.16, pp. 221–225

risk groups. *Human Reproduction*, Vol.6, No.10, pp. 1395–1399

hyperstimulation syndrome in assisted reproductive technology: definition of high

embryo transfer in assisted reproductive technology. *Cochrane Database Systemic*

management of ovarian hyperstimulation syndrome. *British Journal of Obstetrics and*

(1999) Selection of good embryos for transfer depends on embryo cohort size: implications for the 'mild ovarian stimulation' debate. *Human Reproduction*, Vol.14,

ovarian stimulation and endometrial receptivity. *Trends Endocrinology and*

using cleaving embryo grown in vitro. *British Journal of Obstetrics and Gynaecology*,

physiological concepts and clinical consequences. *Endocrine Reviews*, Vol.18, No.1,

cryopreservation programme, present as an optimal choice for MAR.

*Gynaecology*, Vol.102, No,10, pp.767-772

No.12, pp. 3002-8

Vol.87, pp. 737–755

pp. 71–106

*Metabolism*, Vol.15, pp. 84–90

**5. Conclusion**

**6. References**

**4**

*Croatia*

**Prevention and Treatment of Ovarian**

Ovarian hyperstimulation syndrome (OHSS) is a clinical symptom complex associated with ovarian enlargement resulting from exogenous gonadotropin therapy. In severe cases, a critical condition develops with massive ascites, marked ovarian enlargement, pleural effusion, electrolyte imbalance, hypovolemia with hypotension, oliguria, hemoconcentration, and thromboembolism (Madill JJ et al., 2008). Moderate to severe ovarian hyperstimulation syndrome (OHSS) has been calculated to occur in 0.2% to 2% of all ovarian stimulation

Severe OHSS can be a life-threatening complication. The prognosis is usually worse in

The cause of OHSS is unknown, but it may be mediated by vasoactive cytokines secreted in excess by hyperstimulated ovaries (Goldsman MP et al., 1995). Human chorionic gonadotropin (hCG), either exogenous or endogenous (derived from a resulting pregnancy), is believed to be an early contributing factor. Increased vascular permeability is believed to result from vasoactive substances produced by the corpus luteum in response to hCG

The pathophysiology of OHSS is characterized by increased vascular permeability with loss

Gastrointestinal system findings include ascites (third-spacing of fluid), paralytic ileus, and enlarged ovaries. Pulmonary system findings include pleural effusions, restrictive lung disease from ascites or paralytic ileus, and ARDS. Cardiovascular system findings include decreased intravascular volume, decreased blood pressure, decreased central venous perfusion, and compensatory increased heart rate and cardiac output with arterial vasodilation. Coagulation abnormalities include hemoconcentration, increased estrogen level leading to hypercoagulability, and thrombosis. Renal system findings include decreased renal perfusion with subsequent oliguria or renal failure. Hepatic system findings

of fluid, protein, and electrolytes into the peritoneal cavity (Tollan A et al., 1990).

**1. Introduction**

cycles (Binder H et al., 2007).

**2. Pathophysiology**

stimulation (Chen SU et al., 2010).

may include hepatic edema.

patients who get pregnant and have this syndrome.

**Hyperstimulation Syndrome**

Ivan Grbavac, Dejan Ljiljak and Krunoslav Kuna *University Hospital Center "Sisters of Mercy", Zagreb*


*3.1. Granulometry*

Regarding Stage 1 of Figure 1 and using sieves ranging in aperture size from 0.149 to 4.699 mm, the size distribution of the comminuted sample is shown in Figure 2. It can be seen from this figure that nearly 50% of the material passed through the 0.6 mm sieve size. The 80% passing size (d80) of the comminuted mass is about 1.7 mm.

**Figure 2.** Cumulative particle size distribution of the demagnetized and comminuted sample.

#### *3.2. SEM-EDX Analysis*

Image from the Backscatter Electron (BSE) detector on the SEM-EDX demonstrate some dissimilar phases in the demagnetized and comminuted sample (see Stage 1 of Figure 1). "Three Dots" analysis was performed using the EDX. Of the three indicated points shown in Figure 3, each has a distinctive chemistry: point (a) appears to be Ni, which is the outer layer; point (b) is a combination of Pr, Nd and Fe, where Pr is used to substitute Nd (up to 20–25%) to lower production costs; and point (c) shows a mixture of Nd and Fe. It is noted that boron (B) was not detected due to its low intensity, resulting from it being a light element (i.e., element #5 on the periodic table), as well as from the inherent disability of the BSE detector that was used [45].

**Figure 3.** SEM image and EDX composition of the demagnetized and comminuted sample (see text for details). (**a**) Ni; (**b**) Pr, Nd, Fe; (**c**) Nd, Fe.

#### *3.3. XRD Results*

Figures 4 and 5 show the phase compositions of the precipitates (Stage 2 feed to Stage 3 rich, dry precipitate, as per Figure 1) and the final product (Stage 3 REF residue, as per Figure 1), respectively. From Figure 4, it can be said that most of the Fe remains in solution and that the dry precipitate is made up of mainly (NH4)Nd(SO4)2(H2O)3. This compound is an ammonium neodymium sulfate double salt and is noted to be similar to ammonium jarosite [17] with Nd substituting for Fe. Figure 5 also shows the phases of the product made after adding and stirring the dry precipitate in NH4F·HF for 45 min, which is then heated under argon atmosphere to 400 ◦C and allowed to cool. Equation (4) depicts a possible mechanism for REF formation with the REE being Nd assuming ammonium (NH4+) is in excess:

$$2\text{(NH}\_4\text{)Nd(SO}\_4\text{)}\_2\text{(H}\_2\text{O)}\_3 + 3\text{NH}\_4\text{F-HF} + 3\text{NH}\_4^+ \rightarrow 2\text{NdF}\_3 + 4\text{(NH}\_4\text{)}\_2\text{SO}\_4 + 6\text{H}\_2\text{O} + 3\text{H}^+ \tag{4}$$

The product is clearly a REF containing REEs of Nd, Dy and Pr. It can therefore be concluded that REF can be produced by this process and the resulting fluorides can be used later as feedstock for pyrometallurgical metal production [39].

**Figure 5.** Crystal phases in the REF filtrate after 45 min in NH4F·HF and heated to 400 ◦C.

#### *3.4. ICP Results*

Table 1 shows the chemical composition of REEs in the final REF product indicating Dy and Fe are present, apparently in amounts below the XRD detection limit. Together, the total REE amount sums to 63.25%. Based on stoichiometry and assuming the REEs exist as REF (i.e., NdF3, PrF3 and DyF3), the F-content would be 24.94%. This leaves a balance of 10.59%, which is expected to be ammonium sulfate [(NH4)2SO4] and ammonium jarosite [(NH4)Fe3(SO4)2(OH)6], as per Equation (4). Furthermore, based on the stoichiometry of the jarosite, the Fe content would equate to 3.44% of these other components, suggesting there is 7.15% ammonium sulfate. Both ammonium sulfate and ammonium jarosite can be monohydrated [46] which will thermally decompose at low temperature. In this regard, a TGA-DSC study was undertaken on the final REF residue from Stage 3. Because the product was heated to 400 ◦C under argon, a subsequent TGA-DSC scan did not exceed 400 ◦C. It is important to note that this REF residue, as discussed above, is not pure, and therefore contains some ammonium sulfate and ammonium jarosite.

**Table 1.** The chemical composition of the final product.

#### *3.5. TGA-DSC Results*

The TGA-DSC graphs of the REF residue are shown in Figure 6. The plot shows that mass loss occurs in five steps which agrees with the literature [46–49]. In this regard, the first two steps equate simply to crystalline water loss of the ammonium sulfate (Equation (5)) and ammonium jarosite (Equation (7)), respectively:

25–50 ◦C

$$\text{(NH}\_4\text{)}\_2\text{SO}\_4\cdot\text{H}\_2\text{O} \rightarrow \text{(NH}\_4\text{)}\_2\text{SO}\_4 + \text{H}\_2\text{O} \tag{5}$$

75–125 ◦C

$$\text{(NH}\_4\text{)Fe}\_3\text{(SO}\_4\text{)}\_2\text{(OH)}\_6\text{-H}\_2\text{O} \rightarrow \text{(NH}\_4\text{)Fe}\_3\text{(SO}\_4\text{)}\_2\text{(OH)}\_6 + \text{H}\_2\text{O} \tag{6}$$

The third step corresponds to the decomposition of dehydrated ammonium sulfate to ammonium bisulfate:

140–210 ◦C

$$\text{N(NH}\_4\text{)}\_2\text{SO}\_4 \to \text{NH}\_4\text{HSO}\_4 + \text{NH}\_3\tag{7}$$

The fourth step is likely caused by complete thermal decomposition of the ammonium bisulfate: 220–250 ◦C

$$\text{NH}\_4\text{HSO}\_4 \rightarrow \text{1/3NH}\_3 + \text{1/3N}\_2 + \text{SO}\_2 + 2\text{H}\_2\text{O} \tag{8}$$

Finally, the fifth step appears to be the dehydroxylation of the dehydrated ammonium jarosite, thereby accounting for the continued loss in weight as the temperature increased to 380 ◦C:

>260 ◦C

$$(\text{NH}\_4)\text{Fe}\_3(\text{SO}\_4)\_2(\text{OH})\_6 \to (\text{NH}\_4)(\text{FeO})\_3(\text{SO}\_4)\_2 + 3\text{H}\_2\text{O} \tag{9}$$

If the temperature had been increased to 600 ◦C, three additional steps involving the sequential reactions of the dehydroxylated ammonium jarosite would be observed such that all of the Fe ultimately becomes hematite [46–49]:

>385 ◦C

$$2\text{(NH}\_4\text{)(FeO)}\_3\text{(SO}\_4\text{)}\_2 \to 2\text{NH}\_3 + \text{H}\_2\text{O} + 2\text{Fe}\_3\text{O}\_{2.5}\text{(SO}\_4\text{)}\_2\tag{10}$$

>510 ◦C

$$2\text{Fe}\_3\text{O}\_{2.5}\text{(SO}\_4\text{)}\_2 \rightarrow 2\text{Fe}\_2\text{O}\_3 + \text{Fe}\_2\text{(SO}\_4\text{)}\_3 + \text{SO}\_3\tag{11}$$

>540 ◦C

$$\text{Fe}\_2\text{(SO}\_4\text{)}\_3 \rightarrow \text{Fe}\_2\text{O}\_3 + 3\text{SO}\_3 \tag{12}$$

where Fe3O2.5(SO4)2 is essentially equivalent to a solid solution of 2/3 Fe2(SO4)3 and 5/6 Fe2O3.

**Figure 6.** TGA-DSC curve for REF residue from Stage 3.

#### **4. Model Development and Process Optimization Using RSM**

Table 2 shows the Design of Experiments (DOE) along with the condition variables and responses for Stage 3 processing. In all, 17 experiments were performed, each using 2 g of REE-rich dry precipitate from Stage 2. The following ranges of the condition variables were employed: deionized water volume was 10–30 mL; 2–4 g of NH4F·HF was added; the degree of stirring was 15–45 min. Responses were REE recovery and REF purity where recovery refers to the amount of REE from Stage 2 being converted to REF residue and purity refers to the quality of the REF residue based on ICP analysis.

Furthermore, five experiments were completed with all condition variables being at their midpoints. The results were analyzed to develop a statistically significant model for the responses of the REE recovery and REF purity as shown in Equations (13) and (14):

$$R \to \text{RCE coverage } (\%) = \\$0.14 - 3.87 \text{A} + 0.087 \text{B} + 0.84 \text{C} - 2.70 \text{AB} - 0.53 \text{AC} + 2.68 \text{A}^2 + 4.15 \text{C}^2 + 4.04 \text{A}^2 \text{B} + 1.41 \text{A}^2 \text{C} \tag{13}$$

$$\text{REF\\_Purity} \left( \% \right) = 54.75 + 4.20 \text{A} + 1.65 \text{B} - 1.06 \text{C} - 1.59 \text{AB} + 4.37 \text{AC} + 0.18 \text{A}^2 - 1.40 \text{B}^2 - 2.08 \text{C}^2 - 1.17 \text{A}^2 \text{B} + 2.89 \text{A}^2 \text{C} \quad \text{(14)}$$

where A denotes the volume of deionized water used, B denotes the amount of NH4F·HF added, and C denotes the degree of stirring. In both cases, a cubic model represented the data best and the *R*2 values were 0.94 and 0.93 for recovery and purity, respectively.

Using Equations (13) and (14), 3-D surface plots in Figures 7 and 8 were generated to illustrate the e ffects of the condition variables on the responses. From Figure 7a,b, it can be said that NH4F·HF addition appears to have more prominent e ffect on the process e fficiency. The impact of deionized water addition is more prominent at higher stirring rates and higher NH4F·HF additions. With respect to stirring, it can be observed from Figure 7a that the amount of REF recovery increases with stirring time for both 10 and 30 mL of water used. However, recovery is higher for lower volumes of water than for higher ones, and this same pattern is observed with respect to NH4F·HF addition as illustrated in Figure 7b when using Equation (13).

**Figure 7.** 3D plots of REF recovery with respect to process variables at (**a**) 4 g of NH4F·HF and (**b**) 43 min for degree of stirring.

**Figure 8.** 3D plots of REF purity with respect to process variables at (**a**) 4 g of NH4F·HF and (**b**) 44 min for degree of stirring.

Figure 8a,b were generated from Equation (14) and illustrates the response surfaces for REF purity in relations to the process variables. From these plots, it is observed that the amount of deionized water again did influence purity of REF recovery to a significant extent at all NH4F·HF additions and also at higher stirring degrees. The degree of stirring, as observed in Figure 8a, seems to affect the REF insignificantly at low water additions. However, REF purity increased sharply with stirring time at a high volume of water addition. Figure 8b shows that NH4F·HF addition has less impact on the purity of REF. Sharp increase in the purity of the REF is seen with increase in the volume of water.

It may also be noted that, in Equations (13) and (14), the process variables are all in terms of the coded values and differ in the range +1 to −1, with the mid-point having a value of zero. Thus, for B, the maximum amount of NH4F·HF used (4 g) corresponds to +1 and the lowest (2 g) corresponds to –1, whereas the midpoint of 3 g corresponds to 0. To use these statistically significant equations, the actual variable value needs to be converted to the coded form lying between +1 and −1. Using the above model equations optimization was carried out to find the conditions that maximized both REF recovery and REF purity. The identified conditions and their maximum corresponding responses are as follows: