Evaluating Factors Affecting the Success of Embryo Transfer Procedure of the Preimplantation Embryo in IVF

NATIONAL AND KAPODISTRIAN UNIVERSITY OF ATHENS SCHOOL OF HEALTH SCIENCES DEPARTMENT OF MEDICINE PHYSIOLOGY LABORATORY

MSc. MOLECULAR AND APPLIED PHYSIOLOGY

Evaluating factors affecting the success of embryo transfer procedure of the preimplantation embryo in IVF

ROUNGOU PINELOPI-CHRISTINA

Biologist

R. N. 20160442

ATHENS

OCTOBER 2018

SUPERVISING COMMITTEE:

Simopoulou Maria Assistant Professor, Physiology Laboratory, Department of Medicine (supervisor)

Koutsilieris Michael Professor & Head of Physiology Laboratory, Department of Medicine

Philippou Anastassios Assistant Professor, Physiology Laboratory, Department of Medicine

Contents

Prologue ...... 5 Abstract ...... 6 Dissertation purpose ...... 7 1 Introduction ...... 8 1.1 Assisted Reproductive Technology ...... 8 1.2 In vitro fertilization ...... 12 2 Importance of Embryo Selection prior to transfer ...... 16 2.1 Day 0: Oocyte / Pre-zygote quality ...... 17 2.2 Day 1: Two-cell embryo (Zygote) quality ...... 17 2.2.1 Pronuclear Scoring System ...... 18 2.2.2 Cytoplasmic Appearance at Pronuclear Stage...... 22 2.2.3 Time of First Cleavage ...... 23 2.3 Day 2: Four-cell embryo quality ...... 24 2.4 Day 3: Eight-cell embryo quality ...... 25 2.5 Day 4-5-6: Morula & Blastocyst quality ...... 27 2.6 SART Grading System ...... 29 3 Embryo Transfer (ET) ...... 32 3.1 Preparation of the patient ...... 32 3.2 Preparation of the catheter ...... 35 3.3 Transfer of the embryos ...... 37 3.4 Care of the patient ...... 38 4 Variables affecting a successful Embryo Transfer ...... 40 4.1 Embryo Quality ...... 40 4.1.1 Developmental stage ...... 40 4.1.2 Frozen versus fresh embryos ...... 41 4.2 Physiological and anatomical factors ...... 42 4.2.1 Anatomy of cervix and uterus ...... 42 4.2.2 Endometrial receptivity and endometriosis ...... 45 4.2.3 Cervical stiffness and stenosis ...... 47 4.2.4 Psychological parameters and mental state ...... 48 4.3 Technical approaches ...... 48 4.3.1 Catheters and Syringes ...... 48

4.3.2 Ultrasound guidance ...... 54 4.3.3 Types of embryo loading and volume (catheter wash and check - reload) .... 56 4.3.4 Types of media employed for ET ...... 57 4.4 Overall Transfer procedure - performance ...... 58 4.4.1 Number of embryos transferred ...... 58 4.4.2 Uterus position ...... 59 4.4.3 Presence of air in catheter ...... 60 4.4.4 Fluid dynamics and pressure ...... 60 4.4.5 Transfer depth of embryo placement ...... 62 4.5 Observations during ET procedure and their importance (mucus, blood, comfort, reload, use of tenaculum / obturator) ...... 62 4.5.1 Presence of mucus and blood in catheter ...... 62 4.5.2 Difficulties during transfer process ...... 63 4.5.3 Reload phenomenon ...... 64 4.5.4 Uterine contractions by use of tenaculum / obturator ...... 65 4.5.5 Patient’s bed mobilization ...... 66 5 Success rates following Embryo Transfer ...... 67 5.1 Parental age ...... 67 5.2 Frozen versus fresh embryos ...... 67 5.3 Embryo developmental stage ...... 68 5.4 Loading technique ...... 69 5.5 Presence of air bubble in uterus ...... 70 5.6 Mobilization and bed rest after Embryo Transfer ...... 71 5.7 Healthy or ectopic pregnancy...... 72 6 Discussion ...... 73 6.1 Latest trends in Embryo Transfer ...... 73 6.2 Optimal practice and the need for a universal protocol ...... 76 6.3 Future prospects...... 81 6.4 Conclusions ...... 84 Summary ...... 86 References ...... 90

Prologue

This thesis dissertation has been elaborated during my studies of the postgraduate program “Molecular and Applied Physiology” in the department of Medicine at National and Kapodistrian University of Athens. It was conducted in the laboratory of Physiology in 2018. First of all, I would like to express my gratitude to my supervisor, Assistant Professor Maria Simopoulou for the assignment of the present thesis, the valuable opportunity she gave me to collaborate with her as well as her support during this project. I particularly thank her PhD student Anna Rapani for her valuable help that she provided to me at all stages of this dissertation until it was completed. I would like to express my gratitude to Professor Michael Koutsilieris and Assistant Professor Anastassios Philippou for participating as members of the thesis committee. Finally, through the depths of my heart I want to thank my parents, my family and my friends for their love, constant support and encouragement in continuing my studies and fulfilling my dreams and goals.

Abstract

Embryo transfer (ET) is a critical step in the success of in vitro fertilization, where embryos generated into a laboratory are placed in the uterus. The aim of ET is to gently locate the embryos to the endometrial cavity, to a location where implantation has more probabilities to occur. Over the past 30 years the procedure of ET has remained nearly unchanged. Despite the major improvements in embryo culture, genetic screening, embryo selection and embryo cryopreservation, physicians and embryologists often ignore various ET variables which might impact implantation and clinical pregnancy rates and underestimate transfer importance on an IVF cycle. Several variables associated with ET have been investigated in an effort to minimize such issues and increase the efficiency of this clinical practice in the IVF process.

Although ease and safe transfer has been and still remains the topic of interest, it has become clear that reproductive research industry lacks tangible evidence on an overall approach of this process. Thus, future investigations should be conducted in order to examine and evaluate important ET aspects as well as various technical trends namely endometrial scratching, hCG injection, adjutant compounds and biomarkers. In an effort to identify the underlying elements and weighing factors affecting ET procedure, promising data have been surfaced. This valuable information might assist towards achieving a successful IVF-ET treatment, a healthy ongoing pregnancy and ultimately a delivery of a healthy baby.

Dissertation purpose

Over the past years, a revolution of technologies in assisted reproduction (ART) can be observed that has ultimately improved implantation rates. Refinements mainly in embryo selection criteria have improved routine production of viable and good quality embryos “in vitro” in order to achieve clinical pregnancy and result to the birth of healthy offsprings. In vitro fertilization (IVF) is the procedure in which fertilization of an oocyte is performed in a laboratory environment with the purpose of forming a zygote. The fertilized egg is cultured under strict protocols and the best quality-accessed embryos are transferred to the woman's uterus.

The ET process is assessed by important protocol steps and technical details: the embryo selection criteria about embryo quality (morphology as well as frozen or fresh embryos), patient’s clinical state (endometrial receptiveness and reproductive issues), types of ET catheters and syringes, the use of ultrasound guidance, the right position of embryos in the ET catheter, the type of loading, the type of culture media and the appropriate volume, “washing” or not the catheter of choice and by which method, the gentle embryo insertion into the uterus and the examination of catheter following ET for retained embryos (reload phenomenon), blood and mucus. Following procedure, complications may lead to an endometrial trauma and induction of uterine contractions due to release of prostaglandins (PGs). These circumstances could damage the embryos or provoke a deposition of embryos in a location of minimum implantation probability.

Despite revolutionary improvements, the process of embryo transfer (ET), the final step of IVF process, has remained unchanged. Embryos of either day two, day three or day five are transferred in the uterine cavity through the cervical canal. The aim of ET is to gently place the embryos in the endometrial cavity. Various ET variables which might impact pregnancy rates have been underestimated by physicians and embryologists for a long time. Nowadays numerous technical aspects of this method have been studied to minimize complications of this procedure and determine their effect on clinical pregnancy rates. Moreover, there is evidence that certain methods in the ET are associated with improved outcomes following IVF. Thus, the current thesis will review various ET techniques performed during an IVF cycle, variables affecting ET success and approaches of optimization. We will take into account important aspects of physiology of reproduction along with the physiology of the preimplantation embryo and perform a critical analysis of the current literature in order to identify and present the underlying elements and weighing factors in affecting ET procedure outcomes and success rates.

1 Introduction

1.1 Assisted Reproductive Technology

In most western societies, fertility rates have declined over the past years. Various factors are responsible for this decline including the interest in advanced education and career development, marriage at an advanced, delayed childbearing, enhanced contraceptive methods and increase of pathological incidents hindering consequences to an infertility extent (Image 1.1.1).

Image 1.1.1: Common reasons for infertility (Morales, 2011).

Despite the dramatically declined fertility rates over the past decades, technology has been evolved and thrived in many fields. Thus, the application of new technologies in treating human infertility has contributed to the development of Assisted Reproductive Technology (ART), an innovation employed to achieve pregnancy artificially. In circumstances where pregnancy is medically impossible or jeopardizes woman’s health, ART includes procedures involving the manipulation of gametes and embryos outside the human body to achieve fertilization. In case of genetic counseling, ART is also implemented in cases of preimplantation diagnosis for fertile couples diagnosed with some genetic inheriting disorders (Rosenwaks & Wassarman, 2014).

ART includes a wide spectrum of technologies such as infertility treatment, in vitro fertilization (IVF) and surrogacy arrangement (Gardner, Weissman, Howles, & Shoham, 2009; Nagy, Varghese, & Agarwal, 2012; Strauss & Barbieri, 2009). Less invasive methods include ovulation induction (OI), intrauterine insemination (IUI) or artificial insemination (AI) and donor treatment. Advanced invasive techniques involve in vitro fertilization (IVF),

intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD). These techniques could be described in Table 1.1.1 and Image 1.1.2.

Table 1.1.1: Types of Assisted Reproductive Technologies.

Assisted Reproductive Technology (ART) Treatment options Description

 Less invasive techniques

OI may be employed for women who are not ovulating or are not ovulating regularly. They could be under a hormone medication which stimulates the oocyte production. Ovulation induction (OI) This encourages the development of one or more follicles. When the follicles are large enough, hormone human chorionic gonadotropin (hCG) is administered and then the oocytes are released from the follicles.

IUI/AI is employed to help women who have normal, healthy and patent fallopian tubes, but for unknown reasons fail to conceive naturally. During this process prepared semen is inserted through Intrauterine insemination (IUI) the cervix and into the uterus during the ovulation phase. AI can or Artificial insemination (AI) be performed during a natural menstrual cycle or in combination with ovulation induction (OI) (in case of irregular menstrual cycles).

Donor sperm, eggs or embryos could be employed in ART.

 Donor sperm donation or donor insemination (DI) may be used when: (i) a male partner has low sperm count or mobility, (ii) his sperm is not normal, or (iii) there is a high risk of passing on a genetic disease or abnormality to his offspring. The process of DI is similar to intrauterine insemination (IUI).

 Donor eggs treatment is a possible option when: Donor treatment (i) a woman is unable to produce oocytes or her oocytes are characterized as low quality (due to age or premature ovarian failure), (ii) she has reported several miscarriages in the past, or (iii) there is a high risk of the inheriting genetic disorder or abnormality to her offspring. When the oocytes are mature, they are retrieved and then fertilized by sperm. The embryos developed are inserted into the uterus.

 Donor embryos could be implemented if a person or couple requires donor sperm and donor eggs to achieve a pregnancy.

 Advanced invasive techniques

IVF is employed to assist conception and pregnancy especially for women whose fallopian tubes are damaged. In IVF the oocytes and sperm are collected. They are left in a culture dish in the In vitro fertilization (IVF) laboratory to allow the oocytes to be fertilized. If fertilization occurs and an embryo develops, the embryo is placed into the uterus in a procedure called embryo transfer (ET). Sometimes multiple embryos may develop.

GIFT could be described as a natural alternative of IVF. Instead of fertilization occurring in a culture dish in a laboratory, the oocytes Gamete intrafallopian transfer are retrieved from the ovaries and inserted between two layers (GIFT) of sperm. Then they are placed into a fallopian tube where they are left to fertilize naturally. Nowadays GIFT is very rarely used.

ZIFT may be employed by women whose fallopian tubes are Zygote intrafallopian transfer blocked. During the procedure, oocytes are collected from the (ZIFT) ovaries and in vitro fertilized by sperm. The developed embryos are placed into the fallopian tube.

ICSI is employed for the same circumstances as IVF. Oocytes are aspirated from the ovaries. Every oocyte is captured with a specialized holding tool-pipette. A very delicate, sharp and hollow Intracytoplasmic sperm injection needle is used to immobilize and select a single sperm and then it (ICSI) is carefully inserted into the cytoplasm. The sperm is injected and the needle is carefully retracted. The direct injection of a single sperm into each oocyte enhances the possibility of achieving fertilization.

PGD is employed in cases of inherited genetic disease or chromosomal abnormality. PGD could be performed for: (i) couples who have a family history of a genetic disorder or a chromosomal abnormality with high risk of passing on to their children, (ii) couples who had repeated miscarriages or IVF failures in their Preimplantation genetic reproductive history, and diagnosis (PGD) (iii) women of advanced age.

Embryos are developed through the process of IVF or ICSI and then one or two cells are removed from the embryo and screened for a genetic condition. Healthy embryos, unaffected by a particular genetic disease, are selected for transfer to the uterus.

Surrogacy is the method in which a woman (the surrogate  Surrogacy mother) carries an embryo for another person or couple.

Image 1.1.2: Major types of ART (modified) (Neo-Est Scandinavian Fertility Centre, 2014).

IVF is probably the most well known and most widely applied ART procedure. Without IVF, the pregnancy rates in cases of untreatable infertility would remain diminished. For several decades this method has provided infertile couples with the chance to conceive and bear children.

1.2 In vitro fertilization

IVF constitutes the base of infertility treatment when other approaches of ART have failed. This technique has evolved as a routine procedure and it is widely employed all over the world. During an IVF cycle, the fertilization of an oocyte (also known as egg, ovum) by a sperm takes place outside the body, “in vitro” in a laboratory. The fertilized oocyte (zygote) is cultivated and the embryo is transferred to the woman's uterus in order to establish a successful pregnancy and to result in a healthy offspring.

IVF is typically implemented following continuously failed attempts of conception and other types of fertility treatments which were not successful. IVF may treat a wide range of fertility problems caused by a number of reasons including female ovulation disorders such as polycystic ovary syndrome (PCOS), premature ovarian failure, uterine fibroids, genetic disorders, blocked or damaged or removed fallopian tubes and also the male infertility such as decreased sperm count or mobility (Quigley & Wolf, 1984).

In reproductive science, embryo development begins with fertilization (Wu, 2012). Prior to the procedure of fertilization in a laboratory environment, there are some crucial steps that determinate the success of IVF process. For example, gametes must be obtained outside human body and undergo a series of events in order to achieve fertilization. In addition, following fertilization, there are equally important stages, such as the embryo transfer, that are critical to establish a successful healthy pregnancy (Hua et al., 2016; Nagy et al., 2012; Strauss & Barbieri, 2009). In particular, IVF is a four-stage procedure that is described below according to Images 1.2.1, 1.2.2 (a) and (b) and 1.2.3 as well as Table 1.2.1:

Image 1.2.1: In vitro fertilization stages (Rocky Mountain Fertility Center, n.d.).

(a) (b)

Images 1.2.2 (a) and (b): Mechanism of action of gonadotropins (Gn) in the female reproductive system. (a): The gonadotropins’ family includes the follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) which are part of a complex endocrine pathway (modified) (Utiger, n.d.). (b): The hypothalamic-pituitary-ovarian axis, (+) represents positive feedback and (-) negative feedback. They regulate growth, sexual development and normal function of the reproductive system. LH and FSH are secreted by a part of the brain, the anterior pituitary gland. hCG is secreted by the placenta in pregnant women. The gonadotropins act on the gonads by controlling gamete and sex hormone production. They are usually used for infertility therapy (J. Sun, Zhang, Wang, & Yan, 2013).

Image 1.2.3: An idealized example of menstrual cycle where ovulation occurs on day 14 and the entire cycle lasts 28 days. The menstrual cycle comprises parallel ovarian and endometrial cycles. In the follicular phase the menses start on day 0 and new follicles in the ovary mature. The main hormone of this stage is estradiol. It ends with ovulation. The luteal phase begins with the formation of the corpus luteum and ends in either pregnancy or luteolysis (the corpus luteum degenerates). Significant characteristic of this phase is very high levels of progesterone (Boron & Boulpaep, 2017).

Table 1.2.1: The four stages of an IVF procedure.

In vitro fertilization (IVF) Major Stages Sub-stages Description

Stimulation of ovaries is the method to induce the production of follicles. Women are injected with shots of follicle- stimulating hormone (FSH) and are checked by monitoring their estradiol levels with diagnostic tests and their follicular growth with the use of ultrasound means. During stimulation A. Hormonal Ovarian it is critical to suppress any ovulation by using a gonadotropin- stimulation stimulation releasing hormone (GnRH) agonist or a GnRH antagonist in order to prevent the endogenous luteinizing hormone (LH) [the mechanism of action of gonadotropins in Images 1.2.2 (a) and (b)]. The last one drug diminishes the risk of ovarian hyperstimulation syndrome (OHSS), which is a life-threatening condition.

When the follicles reached a suitable size, an injection of human chorionic gonadotropin (hCG) is performed to women. hCG acts like the LH (Luteal phase in Image 1.2.3) and Maturation promotes ovulation that would occur approximately 2 days induction after hCG administration. But the egg aspiration is performed earlier to ovulation (prior to the fracture of follicles). The oocytes are mature, capable of accomplishing a possible fertilization.

Follicular fluid is transvaginally aspirated from the follicles using an ultrasound-guided needle. The retrieved follicular B. Oocyte fluid is examined in order to identify the eggs. The process is retrieval usually done under mild or or general anesthesia, or extremely rarely in a conscious state

The mature oocytes and sperm are prepared for fertilization Gametes in a procedure called gamete washing. In addition oocytes preparation with high chances of successful pregnancy can be selected from the initial number of identified eggs of the follicular

fluid.

Sperm and oocyte are incubated together in a culture media and fertilization takes place “in vitro”. The egg would be fertilized and two pronuclei are microscopically visible in C. Fertilization the zygote. The fertilized oocyte is transferred to a special growth medium and left for about 2-3 days until the zygote consists of six to eight cells.

Culture of embryos can be performed in an artificial culture medium (which contains numerous components in order to assist and improve embryonic growth) or in an endometrial Embryo culture cell culture (the top cell layer from the woman's own endometrium). The duration of culture is until cleavage or blastocyst stage.

The selection of the best embryos has been mostly based on certain attributes about morphology and cell number. ‘The chosen one’ embryo would need pass through multiple assessments at each developmental stage. In particular the morphological criteria have been shown to have some Embryo quality predictive value for high rate pregnancies. This scoring system and selection studies certain traits about oocyte morphology, appearance of cytoplasm and zona pellucida, polar body, zygote quality (pronuclear scoring system), cleavage and blastomeres. The selected embryos should have a greater chance of implantation in order to achieve a successful pregnancy.

The number of embryos that may be transferred mostly depends on the woman’s age and various health related factors. Embryos are often transferred transvaginally to the D. Embryo uterus through a special thin catheter. Several embryos may transfer (ET) be placed into the uterus to improve rates of implantation and to establish higher rates of clinical pregnancy.

Over the years, improved ovarian stimulation and fertilization processes as well as advanced embryo culture and transfer techniques are becoming commercially available. As a result, better quality embryos may be produced accompanied with high pregnancy rates. In particular Embryo Transfer (ET) method is the final stage of IVF which is conducted to facilitate conception. It holds a critical role in the efficiency of the whole IVF process. The current dissertation topic is to assess parameters and variables associated with ET, providing valuable information for ART and its clinical practice.

2 Importance of Embryo Selection prior to transfer

ET represents the final stage of the IVF process. Although considered as a simple non-surgical intervention, it is a critical step affecting the outcome of IVF. In the early days of IVF, pregnancy rates were poor. The only approach offering increased pregnancy rates was the transfer of large numbers of embryos (Edwards & Steptoe, 1983). Over the last decades implantation rates were improved and the transfer of these large numbers of embryos resulted to increased incidents of multiple high-risk pregnancies. To avoid the complications related to multiple pregnancies, the number of the transferred embryos was decreased. Subsequently, the selection of the embryos with the highest chance of implantation arised as a crucial matter to address during the ET process (Kingsland et al., 1990).

The most widely established method to assess embryo quality has been based on microscopic observations of morphology. The selection of the most adequate embryos has been mostly based on certain attributes regarding appearance and cell number (Sakkas & Gardner, 2005). Some major morphological features are presented in and Image 2.1 and Table 2.1. Traditionally morphological evaluation of embryo quality includes observations with simple microscopy. In the following years, time-lapse microscopy has become available and allowed embryo monitoring accounting time as confounder (Massip & Mulnard, 1980).

Image 2.1: Embryo developmental stages after fertilization (Biomaternity Fertility Specialists, n.d.).

Table 2.1: Timeline of embryo development from pre-zygote to implanting blastocyst (modified) (Strauss & Barbieri, 2009).

Stages of embryo development Days after fertilization Oocyte / egg (Pre-zygote) 0 Two-cell embryo (Zygote) 1 Four-cell embryo 2 Eight-cell embryo 3 Morula 4-5 Blastocyst 5 Implanting, hatched blastocyst 5-6

Embryo development is a dynamic process and practitioners’ evaluation should be quick and precise during the microscopic imaging for monitoring embryos’ progress. Established morphological criteria have been presented to literature as parameters reflecting some predictive value for higher pregnancy rates (Nagy et al., 2012).

2.1 Day 0: Oocyte / Pre-zygote quality

The oocyte quality affects fertilization and subsequent embryo development (Scott, 2003b; Q. Wang & Sun, 2007). An oocyte must have the ability to: (a) resume meiosis in order to produce an ovum (gamete), (b) carry out cleavage after fertilization, (c) develop into a blastocyst, (d) lead to a healthy pregnancy and (e) deliver a healthy offspring (Sirard, Richard, Blondin, & Robert, 2006). Cytoplasmic changes and any dysfunction or dislocation of oocyte components such as meiotic spindle, cell organelles (such as nuclear, mitochondria) or granules can decrease oocyte quality and the subsequent embryo quality (Combelles & Racowsky, 2005; Coticchio et al., 2004).

The use of morphological criteria for the assessment of oocyte quality may not be as precise as the use of egg’s cellular and molecular markers (Rosenwaks, 2017). Although these features help to evaluate and predict the embryo viability in order to achieve a healthy clinical pregnancy. The principal morphological parameters that help assess the embryo developmental fate are presented in Table 2.1.1:

Table 2.1.1: Oocyte’s morphological criteria (modified) (Wassarman, Rosenwaks et al. 2014).

Morphological traits: Observations:  Compactness and thickness of the cumulus oophorus (also called 1. Cumulus-oocyte cumulus cells), complex (COC)  Brightness of the cytoplasm.  Granularity (large or small, homogenous or clustering granules, in center or in periphery of the egg), 2. Cytoplasm  Color,  Location of organelles [nuclear, mitochondria, vacuoles, endoplasmic reticulum (ER)].  Shape (round or ovoid),  Size (large or small), 3. Polar body  Surface (smooth or rough),  Cytoplasm (intact or fragmented).  Thickness, 4. Zona pellucida  Structure.  Size (normal or increased), 5. Perivitelline space  Presence or not of grain. 6. Meiotic spindle  Location.

2.2 Day 1: Two-cell embryo (Zygote) quality

Sixteen to eighteen hours following fertilization, the formation and fusion of pronuclei results in a zygote (with diploid nucleus). Quality-control studies have reported

that major abnormalities of the zygote stage could be detected in pronuclear development (such as unequal sizes of pronuclei, not centrally located in the cytoplasm). This kind of malformations could severely decrease further development of the embryo. Zygotes with unequal pronuclear sizes are associated with a high risk of embryonic anomalies and also mosaicism. Additional changes in the pronuclei may be associated with functional abnormalities in chromosome segregation (Scott, Alvero, Leondires, & Miller, 2000; Tesarik & Greco, 1999).

By microscopic monitoring various observations of morphological parameters, ART scientists have proposed a number of non-invasive scoring systems for zygote stage. Firstly pronuclear scoring system attempt to classify zygotes based on the following characteristics of their two pronuclei: I. symmetry (equal or unequal nucleoli), II. position (in center or at a distance in the cytoplasm), III. location (central or non-central position), IV. alignment.

Other parameters that may be taken into consideration, are cytoplasmic morphology and time of early cell division (cleavage) after oocyte fertilization (Montag, Liebenthron, & Köster, 2011).

2.2.1 Pronuclear Scoring System

Nucleoli in oocytes and zygotes referred to as nucleolus precursor bodies (NPBs), are compact and morphologically different from nucleoli in somatic cells. It has been reported that oocyte NPBs and their alignment are essential for embryonic development. They play an important role in establishing embryo’s axis, an essential phenomenon for embryonic cell place determination. Abnormalities in NPB alignment may potentially have severe consequences in zygote growth and pregnancy progress. In their absence, the eggs complete maturation and can be fertilized, but no nucleoli are formed in the zygotes and embryos, leading to developmental failure. These results indicate that oocyte’s NPBs are essential for development of the embryo (Kyogoku, Kitajima, & Miyano, 2014; Salumets, Hydén- Granskog, Suikkari, Tiitinen, & Tuuri, 2001).

The first and the most important scoring system for assessing pronuclei allows for evaluating several NPBs characteristics (Scott, 2003a; Scott et al., 2000; Scott & Smith, 1998). The zygotes are classified into five groups, each one named as Z-score. The five groups were further divided into four sub-categories (Table 2.2.1.1 and Image 2.2.1.1). Z1- and Z2- scoring zygotes usually are evaluated as better quality embryos and Z3- and Z4- zygotes are considered as embryos of worse quality and lower implantation fate (Nasiri & Eftekhari-Yazdi, 2015).

Table 2.2.1.1: Morphological parameters assessed according to the pronuclear scoring system at zygote stage.

Score: Explanation:

Equal pronuclei (PNs) in number and size, aligned nucleolar precursor bodies (NPBs) at the Z1 pronuclear junction. Z2 Equal PNs in number and size, scattered NPBs.

Equal PNs in number and size, with unequal size NPBs, aligned in one pronucleus at the Z3 pronuclear junction and scattered in the other pronucleus.

Z4 Unequal and distant PNs.

Image 2.2.1.1: NPBs pronuclear scoring system (Scott, 2003a).

Image 2.2.1.2: NPBs grading system (Tesarik & Greco, 1999).

Another main classification of zygotes similar to Scott’s grading system, is based on size, number and distribution of pronuclei or of NPBs (Image 2.2.1.2) (Tesarik & Greco, 1999). Through the years various simplified grading systems have been proposed based on the number, alignment and position of NPBs. One characteristic example of such scoring system is the method reported by Brezinova (Image 2.2.1.3) (Brezinova, Oborna, Svobodova, & Fingerova, 2009). It classifies zygotes into two different patterns ("O" and "Other") depended on pronuclei morphology and an early cleavage rate. Pattern "O" consisted of zygotes with the same number of small or large NPBs distributed in the two pronuclei. Zygotes with non-symmetrical alignments of NPB achieved the "Other" pattern. The second criterion in this assessment is the first mitotic division. Embryos with two blastomeres are classified as early cleavage embryos (EC) and those that do not reach the two-cell stage with intact nuclear membranes are classified as no early cleavage embryos (NEC). EC embryos showed better pregnancy rates compared with NEC embryos. The best outcome for embryo quality using this pronuclear grading system should be an embryo classified as EC and "O" score, resulting in a high chance of pregnancy. Thus, this scoring method for embryo

selection and assessment prior to transfer could be denoted as simple, effective and non- invasive (Nasiri & Eftekhari-Yazdi, 2015).

Image 2.2.1.3: (A) Pronuclear grading system according where the pattern "O" is defined as the same number of small nucleolar precursor bodies (NPBs) distributed in the nucleus or of large NPBs with polar arrangement between the two pronuclei. (B) Pronuclear score where the pattern "Other" characterizes zygotes with non- symmetrical alignments of NPBs (Brezinova et al., 2009).

Recently another method for embryo grading at the two-cell stage has been proposed (Senn et al., 2006). Initially zygotes are graded based on proximity, orientation and centering of the pronuclei, cytoplasmic halo, number and polarization of NPBs. Then the cumulated pronuclear score (CPNS) which is the sum of scores of the six parameters, is calculated for each zygote. CPNS may be a valuable tool in order to predict pregnancy chances for fresh and frozen-thawed zygotes. Frozen-thawed zygotes indicate lower CPNS and this value may indicate that freezing damages zygotes. The grades of NPBs and cytoplasmic halo appear as the most important predictive factor for implantation rate in both types of zygotes. Image 2.2.1.4 shows some examples of zygote scores (1, 2, 3) and CPNS are indicated in parentheses.

Image 2.2.1.4: Examples of zygote scoring. For each zygote, there are scores (1, 2 or 3) for individual feature (proximity, orientation and centering of the pronuclei, cytoplasmic halo, number and polarization of NPBs). The cumulated pronuclear score (CPNS) is indicated in parentheses (Senn et al., 2006).

2.2.2 Cytoplasmic Appearance at Pronuclear Stage

Prior to pronuclear formation, there is a microtubule-mediated withdrawal of cytoplasmic organelles and components from the egg periphery to the center, resulting in a clear halo. Cytoplasmic halo is another important criterion for pronuclear stage embryo grading and is described as a sub-plasmalemmal zone of a translucent cytoplasm. A physiological halo is clear (Image 2.2.2.1). The utility of the halo is still unclear as a marker of embryo quality. Some reported that the halo is linked with increased blastocyst formation and high rates of implantation, while others found that a halo might have a detrimental effect on blastocyst development (Ebner, Moser, Sommergruber, & Tews, 2003; Gardner & Sakkas, 2003). More studies are required before any conclusion reached regarding the suitability of assessing the cytoplasmic halo of a Day-1 embryo. Image 2.2.2.2 illustrates the pronuclear embryo classification based on the presence or not of halo (Depa-Martynow, Jedrzejczak, & Pawelczyk, 2007).

Image 2.2.2.1: A human pronuclear stage zygote (Beuchat et al., 2008).

Image 2.2.2.2: The presence or absence of a cytoplasmic halo: (A) zygote with cytoplasmic halo and (B) zygote without cytoplasmic halo (Depa-Martynow et al., 2007).

2.2.3 Time of First Cleavage

Twenty four to twenty eight hours following fertilization, the first cleavage is present and size, symmetry and fragmentation of blastomeres are assessed. The first cleavage to the two-cell stage has been reported to be one such critical time point for selecting embryos for transfer (Van Montfoort, Dumoulin, Kester, & Evers, 2004).

Early entry into the first mitotic division represents the fulfillment of final events of fertilization (alignment of pronuclei and of chromosomes on the metaphase spindle) and finally the first mitotic division occurs. Also entry into the first cell division has been correlated with increased blastocyst formation, increased implantation rates, increased pregnancy rates and a euploid pair of chromosomes (Giorgetti et al., 2007). Early first cleavage is strongly associated with good embryo morphology on day 1 and demonstrates a valuable predictive value for pregnancy (Montag et al., 2011).

Undoubtedly NPBs pronuclear scoring system is a simple and non-invasive quality- control technique requiring observation of simple morphological traits that enables the scientists to separate zygotes with high implantation potential from those that clearly have a fundamental defect at a molecular and/or cellular level (Rienzi et al., 2005). However, no

standardized scoring system for zygote grading is currently available in ART laboratories. Further refinements of the scoring system are needed to occur in order to allow more accurate assessment of the NPBs, cytoplasm and early cleavage which are indicative of the correlation between embryo quality and implantation fate (Scott, Finn, O’Leary, McLellan, & Hill, 2007).

2.3 Day 2: Four-cell embryo quality

For day 2 embryo scoring systems estimate embryo quality according to the cleavage speed (early cleavage), shape of the blastomeres, and the percentage of fragmentation. Multinucleation, number of blastomeres, cell size and fragmentation on this day are the most notable parameters to predict the blastocyst developmental fate.

Multinucleation is a common phenomenon during IVF cycles where there is more than one nucleus in each blastomere of an embryo. It indicates a breakdown of cellular function and it usually occurs during the first mitotic division due to false chromosome segregation. Multinucleation has been correlated with an increased rate of aneuploidy and chromosomal abnormalities. Multinucleation on day 2 has been linked to poor embryonic development and lower implantation rate (Alikani et al., 2000; Nasiri & Eftekhari-Yazdi, 2015).

In the four-cell stage number of blastomeres holds a critical role into embryo implantation and establishment of a high rate pregnancy. Embryologists have reported that the subsequent blastocyst has higher chances to form from four-cell embryos compared to those with slower cleavage (2–3 cells) or faster cleavage (5–8 cells). On one hand, slower cleaving embryos cannot enter to the blastocyst stage due to their failure to divide on a certain developmental timeline. On the other hand, faster cleaving embryos have blastomeres with large anucleate fragments and higher levels of aneuploidy. Those outcomes indicate serious developmental abnormalities of the embryos (Guerif et al., 2007).

Blastomere size is significant scoring trait. The asymmetry in an embryo with uneven number of blastomeres most likely begins from scattering proteins, mRNAs and various organelles into the cytoplasm between two produced sister cells. This event reflects an asynchrony of cell division. Blastomeres’ unbalanced size has been linked to fragmentation where the degree of fragmentation was correlated with the extent of unequal blastomeres regarding their size. Unqual cell size is associated with poor development potential and implantation rate of embryos (Hardarson, Hanson, Sjögren, & Lundin, 2001; Hnida, Engenheiro, & Ziebe, 2004).

During cell division fragmentation refers to the presence of extracellular cytoplasmic fragments only with abnormalities in nuclear and cytoplasmic cell division, leading to apoptosis or to chromosomal abnormalities because of false segregation. Fragmentation value can be described as a percentage of embryo volume occupied by fragments. A very simple scoring system only for fragmentation is presented in Table 2.3.1. An increased

fragmentation percentage is a significant trait of a low rate blastocyst formation (Hnida & Ziebe, 2004).

Table 2.3.1: Grading system for embryo fragmentation as a criterion.

Scores Percentage of embryo volume occupied by fragments 0 0 % 1 < 10 % 2 10 - 25 % 3 > 25 %

A day 2 embryo grading system evaluates embryo’s blastomeres (Table 2.3.2). The embryos are divided into three different groups based on cell number. Then they are classified by the degree of fragmentation which is expressed as a percentage of the total embryo volume occupied by anucleate cytoplasmic fragments. The presence or absence of multinucleation plays an important role in embryo selection and embryos with multinucleated blastomeres were excluded. Embryos of “top quality” are those with very early cleavage, four regular blastomeres, < 20 % of fragmentation and no multinucleation (Guerif et al., 2007).

Table 2.3.2: Grading systems of 3 morphological features used to assess day 2 embryos.

Cell number Fragmentation score Multinucleation < 4 < 20 % Yes = 4 20 - 50 % No > 4 > 50 %

2.4 Day 3: Eight-cell embryo quality

Commonly embryo quality assessment is performed on the first day (day 1) following egg fertilization. However, 48 hours later (day 2) embryos must have preferably 3 or 4 blastomeres and after 72 hours (day 3) it is expected to observe of preferably 8 cells.

A method for classification of day 3 embryos is developed (Image 2.4.1 and Table 2.4.1) by using the morphological criteria of blastomere number and fragmentation. Embryos are accessed on four grades (A-D) according to the number of blastomeres and the degree of cytoplasmic fragmentation. The best embryos (grade A) have a mean value of 8 blastomeres (7-9 blastomeres) and maximum a 20% of cytoplasmic fragmentation. Also grade B embryos have 7-9 blastomeres and over 20% of fragmentation. Grade C embryos consist of 4-6 cells and have maximum a 20% of fragmentation. Grade D embryos are considered the worst quality (4-6 blastomeres embryos with over 20% of cytoplasmic fragmentation) (Depa-Martynow et al., 2007).

Image 2.4.1: Embryo scoring system based on blastomere number and percentage of fragmentation (Depa- Martynow et al., 2007).

Table 2.4.1: Embryo grading system based on blastomere number and fragmentation (Depa-Martynow et al., 2007).

Grades Explanation A Embryos with 8 blastomeres and > 20% of cytoplasmic fragmentation B Embryos with 8 blastomeres and < 20% of cytoplasmic fragmentation C Embryos with 4-6 blastomeres and = or < 20% of cytoplasmic fragmentation D Embryos with 4-6 blastomeres and > 20% of cytoplasmic fragmentation

An additional system for embryo assessment in cleavage stage (similar to Depa- Martynow’s system) is based on blastomere size and the amount of cytoplasmic fragmentation (Stensen, Tanbo, Storeng, Byholm, & Fèdorcsak, 2010). Table 2.4.2 denotes the scoring methodology. The best quality embryo has equal blastomeres and minimal or no cytoplasmatic fragmentation (score 4).

Table 2.4.2: Morphological grading of embryos based on blastomere size and fragmentation value (Stensen et al., 2010).

Score Description 4 Embryos with equally sized blastomeres and no fragmentation 3 Embryos with even or uneven blastomeres and ≥ 10-20% of cytoplasmic fragmentation 2 Embryos with even or uneven blastomeres and > 20-50% of cytoplasmic fragmentation 1 Fragmentation precluded counting blastomeres 0 No cleavage or morphologically abnormal embryo

Early cleavage plays an important role in quality evaluation before embryo transfer. The optimal embryo for transfer should consist of four blastomeres on day 2 and eight cells on day 3 (Image 2.4.2). It may have less than 20% of fragmentation and the blastomeres must not present multinucleation (multinucleated blastomeres, MNBs) (Pelinck et al., 2010).

Image 2.4.2: Characteristics of embryos scored at day 1, day 2 and day 3 (Gardner et al., 2009).

2.5 Day 4-5-6: Morula & Blastocyst quality

Embryos reach the morula stage on day 4 following fertilization. The morula is characteristically known for the loss of cell borders due to compaction process. Tight junctions are created between cells. This process is important for the formation and transportation of epithelium into the developing blastocyst. Morula embryos are not commonly evaluated. A limited number of morula grading systems have been proposed and used for embryo selection (Feil, Henshaw, & Lane, 2008; Tao et al., 2002). Heavier emphasis has been placed on the day 2 and day 3 scoring systems (Abeyta & Behr, 2014).

Blastocyst formation starts on day 5 following insemination. In that point, blastocyst consists of the inner cell mass (ICM), the blastocoele and the trophoblast. The inner cell mass will form the embryo and is surrounded by a cavity filled with fluid, the blastocoele. The outer layer of the blastocyst consists of cells called trophoblast. Taking advantage of these three layers, Gardner and other scientists estimated embryo’s value and implantation chances (Table 2.5.1 and Image 2.5.1) (Gardner, Lane, Stevens, Schlenker, & Schoolcraft, 2000).

During an IVF-ET cycle a blastocyst transfer could lead to a high pregnancy success rate with very low risk of multiple pregnancies. Many IVF programs that transfer blastocysts, use the blastocyst scoring system which has three separate quality scores for each blastocyst (blastocoele, ICM and trophectoderm) (Table 2.5.1 and Image 2.5.1) (Fragouli, Alfarawati, Spath, & Wells, 2014; Sjöblom, Menezes, Cummins, Mathiyalagan, & Costello, 2006). Blastocoel score is a number from 1-6 based on degree of blastocyst expansion and hatch. The ICM score refers to how many cells are forming the epithelium (A, B, C) and the final TE score describes the cell quantity of trophoblast.

Table 2.5.1: Blastocyst scoring system (Gardner et al., 2000).

Blastocoel Inner cell Trophectoder Explanation: mass (ICM) Explanation: Explanation: score: m (TE) score: score: Early blastocyst: Tightly Blastocoel less 1 A packed, A Many cells than half of many cells blastocyst's volume Blastocyst: Blastocoel Loosely more than 2 B gathered, B Few cells half of few cells blastocyst's volume Blastocyst: Blastocoel Very few 3 equals to C C Very few cells cells blastocyst's volume Expanded blastocyst: Blastocoel is 4 larger in size than the early blastocyst Hatching 5 blastocyst Hatched 6 blastocyst

Image 2.5.1: Blastocyst grading system (Gardner et al., 2000).

2.6 SART Grading System

Nowadays the Society for Assisted Reproductive Technology (SART) has introduced a new method of embryo grading which is simple and comprehensible. According to every embryonic developmental phase (cleavage, morula and blastocyst), the SART method uses 3 grades/scores: Good, Fair and Poor. It makes a categorization of embryos into three groups and distinguishes them depending on the grade scale (Table 2.6.1). In the end this system compiles a list of the most suitable embryos for transfer and implantation (Hossain, Phelps, Agarwal, Sanz, & Mahadevan, 2013).

Table 2.6.1: SART grading system (Heitmann, Hill, Richter, DeCherney, & Widra, 2013; Hossain et al., 2013).

Growth period: Grade: Cleavage Good, Fair, Poor Morula Good, Fair, Poor Blastocyst Good, Fair, Poor

However over recent years there were not adequate high rates of pregnancies by ART (Kupka et al., 2014). It is vital to develop additional or complementary methods that predict the embryo’s viability and obtain valuable information about the quality of embryo

for transfer. Approaches different from the morphological criteria have been suggested and are mostly based on the embryo’s chromosomal status (preimplantation genetic screening) and the embryo’s culture medium spent analyzing proteomics and metabolomics (Rødgaard, Heegaard, & Callesen, 2015; Rosenwaks, 2017; Rosenwaks & Wassarman, 2014).

The evolution of advanced genetic methods enables preimplantation genetic diagnosis (PGD). In several countries the PGD has been introduced in many ART centers. It helps to detect abnormalities and disorders especially in high risk couples. The evaluation of genetic material obtained from embryo blastomeres contributes to detect the most frequent chromosomal abnormalities (such as trisomy of chromosome 21, Down’s syndrome) and gene mutations (such as the genetic condition cystic fibrosis). The fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) are very effective screening tests in PGD (Baczkowski, Kurzawa, & Głabowski, 2004; Gianaroli, Magli, Ferraretti, Fortini, & Grieco, 2003; Scott et al., 2000).

There are also attempts to evaluate embryo culture medium with substances of interest: (i) the proteins translated from the specific gene expression products (mRNAs) (proteomics) and (ii) the end-products of cell biological processes (metabolomics). The proteome represents all proteins translated from the specific gene expression products (mRNAs) of a cell at a specific time, conditions and levels. The embryonic secretome refers to the proteins produced and secreted by the developing embryo. The secretome is changing according to embryo developmental fate and viability (Katz-Jaffe & Gardner, 2008). Unfortunately knowledge of the embryonic proteome and secretome and proteomic technology applications are very limited. However new technological advances have allowed the discovery of potential protein biomarkers (Katz-Jaffe & McReynolds, 2013). Several attempts have been made to establish biomarkers after the analysis of embryonic medium. A potential protein biomarker might be the hormone human chorionic gonadotrophin (HCG) (Butler et al., 2013). HCG has been implemented as an indicator for embryonic development and clinical pregnancy establishment. Although HCG appears to be promising biomarker for prediction of embryo transfer success, it was not possible to be detectable in the culture medium prior to blastocyst hatching stage (day 6). Another controversial example is human leukocyte antigen-G (HLA-G) which is thought to play an important role in embryo implantation and maternal tolerance of fetal tissue (Lundin & Ahlström, 2015). The validity of this single biomarker for prediction of embryo viability remains questionable because of the results of various clinical trials which conclude to the fact that HLA-G does not offer any clinical advantages compared with morphological criteria due to variations in HLA-G contents in embryonic medium between different ART laboratories (Rødgaard et al., 2015).

Metabolomics provide an insight of the concentrations of all metabolites (amino acids, oxidation products, carbohydrates and acids) in the culture medium. Metabolites of a cell (the metabolome) could change according to the metabolism or the environmental condition. Therefore embryo metabolome might be a good indicator of cellular activity, and may present powerful biomarkers for viable embryos having a high probability of implantation success after transfer. During embryo selection procedure based on metabolome, measuring the consumption of oxygen, the uptake of amino acids, pyruvate

and glucose, the secretion of oxidation products and enzymes would allow scientists to evaluate embryos before transfer (Baczkowski et al., 2004; Nagy, Sakkas, & Behr, 2008). However proteomic and metabolomic methods are expensive, not sensitive, time and labor- consuming. Also they are still at initial stages of clinical practice in ART laboratories. This molecular analysis is challenging because proteins and metabolites are available in limited amounts in the embryo culture medium, very difficult to be detected.

Since the start of IVF the selection of embryos has been largely based on morphological characteristics of the embryo. Nowadays alternative methods for embryo selection are. Although due to their minimum clinical use these methods are not reliable to increase pregnancy rates and are not commonly used during embryo evaluation (van Loendersloot, van Wely, van der Veen, Bossuyt, & Repping, 2014). Morphological selection of embryos still remains the “core” of daily laboratory practice in IVF cycles.

3 Embryo Transfer (ET)

Embryo transfer (ET) is a step of pivotal importance for the success of in vitro fertilization, where embryos of either day two, day three or day five are transferred in the uterine cavity through the cervical canal (Image 3.1). The aim of ET is to gently place the embryos in the endometrial cavity. A successful ET should deliver the embryos to an endometrial location where implantation is most likely to occur. Complications may lead to endometrial trauma, induction of uterine contractions, damaged embryos, or deposition of embryos in a suboptimal location. Several ET steps have been examined in an effort to minimize complications and increase transfer efficiency (Aflatoonian & Asgharnia, 2006).

Image 3.1: Embryo transfer (ET), final stage of IVF (“In vitro fertilization (IVF) and its mechanism « gullalaii,” n.d.).

The procedure of ET may be categorized into four sections (Mains & Van Voorhis, 2010; Nagy et al., 2012; Penzias, Bendikson, Butts, Coutifaris, Falcone, et al., 2017; Practice Committee of the American Society for Reproductive Medicine et al., 2017; Rosenwaks & Wassarman, 2014):  Preparation of the patient,  Preparation of the catheter,  Transfer of the embryos, and  Care of the patient.

3.1 Preparation of the patient

1. Days before an IVF cycle a routine uterine cavity evaluation with ultrasound is performed to detect possible uterine abnormalities (leiomyomas, endometriosis and polyps) that may obstruct embryo implantation. In case of abnormalities, surgical removal is recommended to improve ET outcome (Surrey, 2012).

2. Prior to the start of an IVF cycle, an evaluation of the cervical canal and of the uterine cavity’s depth should be performed and be used for guidance during the ET. With ultrasound assessment, the uterine cavity is measured inserting the catheter up to the uterine fundus. The total length of the cervix and the thickness of uterine cavity are calculated (important for clinical touch ET method). The measurements also identify a stenotic or a sinuous cervix and thus a difficult embryo transfer could be estimated. During ET, if inserting a catheter through cervix into the uterine cavity is impossible, it is recommended that the patient could undergo cervical dilation and/or anesthesia.

3. Information regarding the uterine position [anteverted (AV) or retroverted (RV)] is essential to prevent misdirecting the ET catheter along the cervical canal into the endometrial cavity. A trial transfer a few days before the actual procedure could assist to explore the position of the uterus (Sallam, 2015).

4. An ultrasound-guided trial transfer provides with the estimation of the utero-cervical angle, thus avoiding the misdirection of the catheter. This technique ensures an accurate and atraumatic ET procedure.

5. The number of embryos to be transferred should be discussed with the couple and the decision should be based on the clinic’s own data and patient clinical state and age and embryo quality (Practice Committee of the American Society for Reproductive Medicine & Practice Committee of the Society for Assisted Reproductive Technology, 2009).

6. At the day of ET, the patient is placed in an operating bed to a lithotomy position (supine position, legs are bent at the knees and feet are placed in stirrups, Image 3.1.1).

Image 3.1.1: Standard lithotomy position (Wheeless & Roenneburg, n.d.).

7. Anesthesia for embryo transfer is not widely administered. Anesthesia may be employed if the patient has indication of cervical stenosis or a history of difficult embryo transfers.

8. Antibiotics are not routinely administered at the time of ET. In patients who report gynecological diseases and infections in their reproductive history, antibiotics are administered prior to ET.

9. A sterile speculum is placed in the vagina for better visualization of the cervix. A proper cervico-uterine angle may be achieved by the implementation of the speculum.

10. The exterior cervical os is cleaned with sterile gauze moistened with saline or transfer medium. Additional fluid and mucus in the cervix should be removed with an atraumatic catheter such as a soft ET catheter. Mucus may obstruct the delivery of the embryos into the uterus by sticking on the tip of the ET catheter. Embryos may become displaced into a suboptimal endometrial location resulting in low pregnancy rates (Eskandar, Abou- Setta, El-Amin, Almushait, & Sobande, 2007).

11. A trial (dummy) embryo transfer is performed. The trial catheter should be the same type of catheter that will be used for the actual transfer. Differences in trial transfer catheter length, diameter and stiffness compared to the actual ET catheter as well as differences in the patient’s uterine position play a crucial role in the transfer success and the establishment of a healthy pregnancy. A trial ET is important to overcome difficulties that may be encountered in the actual ET. Some common factors that are known to negatively impact the ET, may be (Grygoruk et al., 2012; Spitzer et al., 2012):  presence of blood or mucus on the catheter,  the immediate withdrawal of the catheter,  patient’s cervical stenosis,  no alignment between ET catheter and utero-cervical angle,  stimulation of uterine contractions during ET,  uteri that are bent into a forward and backward direction [anteverted (AV) and retroverted (RV) as well as anteflexed and retroflexed concerning their fundus position (Image 3.1.2).

All difficulties during ET procedure and solutions overcoming them would be discussed extensively in chapter 4. Variables affecting a successful Embryo Transfer.

Image 3.1.2: Uterus positions (Ellis, 2008).

12. The catheter should only be placed 10-15 mm far from the uterine fundus, to an endometrial location for the embryo transfer (Image 3.1.3).

Image 3.1.3: The anatomy of a normal uterus (The Editors of Encyclopaedia Britannica, n.d.).

13. Once the trial transfer is completed, the trial catheter is removed and the embryos are loaded into the actual transfer catheter.

3.2 Preparation of the catheter

There are two types of transfer catheters: Soft and firm. Soft catheters (such as a Wallace catheter, Image 3.2.1) are elastic, bent very easily and follow the natural curves of the cervix and uterus. However, they are challenging to use, especially in patients with extreme utero-cervical angles. Firm catheters [such as a Gyneflex and Rocket catheter, Images 3.2.2 (a) and (b)] present the advantage of being rigid with a curve molded into the tip of the catheter and thus potentially easier to deal with a sinuous cervix. The firm catheters are used only for rare occasions when soft catheters fail to enter the uterine cavity.

Image 3.2.1: A soft catheter (Cook Medical, n.d.-a).

(a)

(b)

Images 3.2.2 (a) and (b): (a) A Gyneflex firm catheter (Adhar Healthcare & Devices (AHCD), n.d.) and (b) a Rocket firm catheter (Rocket Medical, n.d.).

Image 3.2.3: A Wallace soft inner catheter with the outer sheath and the inner catheter (modified) (Nielsen, Lindhard, Loft, Ziebe, & Andersen, 2002).

Some catheters consist of two materials with a soft inner catheter and a firm outer sheath which allow for flexibility and better management during transfer technique (Image 3.2.3). The outer sheath may be gently curved to adjust to cervical curves and align the inner catheter with the cervix axis (such as a Cook catheter, Image 3.2.4). In the majority of cases,

ART laboratories employ soft catheters for embryo transfer procedures that are associated with atraumatic embryo placement to the endometrial lining improving the possibility for implantation.

Image 3.2.4: A Cook catheter (Cook Medical, n.d.-b).

1. The catheter is flushed with embryo culture medium in order to remove all air bubbles from its column.

2. Embryo culture media is employed as a loading fluid. The embryos are aspirated into the catheter with a volume of 20–40 μl of medium so that the embryos are loaded towards the tip of the catheter.

3. The embryos are ready to be transferred and placed into the uterus. During transfer process, ultrasound guidance is required to observe embryo loading into the uterine cavity and also detect any air bubble in the endometrial cavity.

3.3 Transfer of the embryos

ET procedure may be divided into two methods: (1) clinical touch ET and (2) ultrasound-guided ET. Clinical touch ET involves the transfer of embryos into the uterine cavity based on clinical measurements of the cervical canal and the uterus depth prior to ET. The implementation of the trial catheter measurements as a reference to facilitate the transfer catheter position in the uterine cavity has been reported in the literature. This method ensures that the transfer is atraumatic. The embryos are deposited at approximately 10–20 mm before the fundus employing ultrasound means and trial transfer calculations as a guide.

Ultrasound-guided ET represents the transfer of embryos into the uterine cavity at a fixed distance from the fundus while performing ultrasound. Advances in ultrasonographic technology and the use of ultrasound guidance enable to visually track the ET procedure to ensure an accurate and atraumatic deposition of embryos to the implantation site. The bladder should be full in order to cover the uterine fundus for visualizing ET process.

Compared to clinical touch, several studies have reported that ultrasound-guided ET significantly increases the chances of implantation, ongoing pregnancy and live birth. Thus, ultrasound guidance is a useful tool for ET (Brown, Buckingham, Buckett, & Abou-Setta, 2016; Cozzolino et al., 2018; Revelli et al., 2016; Sallam, 2015; Teixeira et al., 2015). Several advantages of ultrasound guidance can be cited: (i) Correct placement of the catheter at a decent distance from the fundus avoiding contact with it, (ii) Assurance of embryo delivery in the approximate implantation site, and (iii) Straightening of the utero-cervical angle in patients with severely bent uterus by full bladder method. Thus, ultrasound could be a valuable tool in managing any difficulty that may occur during transfers.

Disadvantages of ultrasound may include the patient discomfort to a full bladder and pressure from the probe (Brown et al., 2016).

1. The placement of the transfer catheter should be done slowly and gently.

2. The embryos should be located in the mid to lower part of the uterus, avoiding injuring the fundus.

3. After depositing the embryos into the uterine cavity, the syringe should have the same pressure in its column in order to reduce the possibility that the embryos will be aspirated back into the catheter until it is withdrawn from the cervix.

4. The removal of the transfer catheter should be done very slowly and gently.

5. Following transfer, the catheter is passed to the embryologist to be examined for remaining embryos. In that case, embryos should be reloaded into a new transfer catheter and immediately transferred back into the uterus, known as the reload phenomenon.

3.4 Care of the patient

The patients are transferred to a recovery room where they may remain on bed rest for 20-30 min.

The air bubble location following ET is the placement spot of embryos (Image 3.4.1). With ultrasound guidance the site of embryo deposition could be tracked down. Bubble migration can be described as a random movement of the bubbles and possibly the embryos (towards the fundus, bubble motion towards the cervix is uncommon), even with the patient in the horizontal position following the procedure. However, it should be noted that bed rest may not be necessary. Similar birth rates were observed in patients with air bubbles moving towards the fundus compared with those where air bubbles remained stable following transfer (Spitzer et al., 2012; Tiras et al., 2012a).

Image 3.4.1: Placement spot of air bubble pointed by the red arrow (modified) (Allahbadia & Chillik, 2015).

Image 3.4.2: Summary of overall Embryo Transfer (ET) process (modified) (Siam Fertility Clinic, n.d.).

4 Variables affecting a successful Embryo Transfer

4.1 Embryo Quality

4.1.1 Developmental stage

Until recently it was widely accepted that in order to achieve high pregnancy rates, two or more embryos had to be transferred. However, in order to reduce the multiple birth rate, the number of transferred embryos had to be reduced. Single embryo transfers are now considered a routine practice in many ART laboratories. The importance of selecting the single most viable embryo for transfer has intensified the search for improving the assessment of embryo quality.

In IVF programs human embryos usually are transferred at the blastocyst stage (Glujovsky & Farquhar, 2016; Glujovsky, Farquhar, Quinteiro Retamar, Alvarez Sedo, & Blake, 2016). However, there are studies in which embryos have been transferred at the cleavage stage. This developmental of stage has been claimed to allow a high percentage of embryos to develop to blastocysts with a high viability and implantation rate. But there are two main reasons why an alternative embryo transfer stage was proposed. Firstly it has been recognized that the exposure of early-stage embryos to the uterine environment is too premature. In vivo, embryos travel through the fallopian tubes and do not reach the uterus before the morula stage. The uterus provides a different nutritional environment from the fallopian tube. This phenomenon may cause stress on the embryo resulting in a reduced implantation potential. Secondly there are widely acknowledged outcomes of the morphological criteria used for selection of cleavage-stage embryos for transfer. Prior to day 3 after fertilization, when genome activation and compaction begins, embryo development is controlled by maternal-origin RNA messages. Only after this stage, the development is under the control of an activated embryonic genome resulting in the expression of numerous growth factors and receptors. Furthermore it is suspected that before day 3 cleavage-stage embryos are chromosomally abnormal with high rate of implantation failure. Extending embryo culture until the blastocyst stage might provide advantages by allowing transfer of embryos into the uterine environment and by selecting only those embryos that have demonstrated the potential for clinical pregnancy (Johnson, Blake, & Farquhar, 2007).

To increase successful implantation rates and reduce the potential for multiple pregnancies, early cleavage should be assessed very carefully before viable embryos and blastocysts are selected for transfer. Because blastocyst formation may be influenced by long-term culture, air, and other culture conditions, patients with fewer oocytes or embryos should receive transfer of viable day 3 embryos with well-defined early cleavage rather than on transfer of viable embryos with unclear early cleavage. This maximizes the probability of pregnancy (C.-I. Lee et al., 2016; Maheshwari, Hamilton, & Bhattacharya, 2016). In addition, morula embryo transfer provides success probabilities similar to blastocyst embryo transfer. If good morula-stage embryos are transferred, their pregnancy rates could be compatible

with those of good blastocysts, thus providing time flexibility for transfer process (R.-S. Li, Hwu, Lee, Li, & Lin, 2018).

4.1.2 Frozen versus fresh embryos

Cryopreservation of human gametes and embryos is an important technique in ART that leads to increased pregnancy outcomes (Youssry, Ozmen, Zohni, Diedrich, & Al-Hasani, 2008). This approach limits the number of embryos transferred while extra oocytes and/or embryos can be used in subsequent treatment cycles. Cryopreservation is carried out with two techniques: the slow-freezing and the vitrification method.

In slow-freezing cryopreservation human gametes and embryos are preserved in liquid nitrogen. There are five stages in this procedure: (1) Initial exposure to cryoprotectants, substances which reduce cellular damage caused by the crystallization of water (gametes and embryos consist of water in their cytoplasm), (2) Freezing to temperatures below 0°C, (3) Storage, (4) Thawing, and (5) Dilution and removal of the cryoprotectants and the return to a physiologic cellular function.

The most critical moments for cellular survival and development are the initial phase of freezing at a very low temperature and the final return to physiologic conditions. If a sufficiently low temperature is reached (normally −196°C in liquid nitrogen), even for a long period of time, has no effect on the survival rates of gametes and embryos.

Although cryopreservation is a fertility solution for patients with iatrogenic sterility (after chemo/radiotherapy in cancer diseases) and for women who suffer from pathologies of the reproductive system suppressing the function of the ovaries (premature ovarian failure, endometriosis, cysts, and pelvic infections), it is known that following slow-freezing cryopreservation is the intracellular ice crystal formation that generally pierces the membrane. This causes lysis and breaks the meiotic spindle resulting in chromosome aneuploidy. Because the human gametes and embryos consist of water in the cytoplasm, it is difficult to avoid ice crystal formation with the slow-freezing protocol. Vitrification is a new, simple, rapid process that produces a glass-like solidification of living cells which completely avoids ice crystallization during cooling and warming (Gardner et al., 2009).

With each technique, human gametes and embryos can be preserved in very low temperatures and can be used in future infertility treatments in ART programs.

Frozen–thawed embryo transfer (FET) is the most common way to increase pregnancy rate. It has been shown that the same number of oocytes is needed in fresh and vitrified cycles conclude to similar results in terms of embryo development and quality. Also, the rate of hCG rise following FET is significantly higher than fresh ET hCG rate, but FET and

fresh ET conclude to the same live birth rates (Slomski, 2018). Some studies can support that higher embryo implantation and clinical pregnancy rates are achieved in FET as well as in fresh ET especially in patients with a strong ovarian response [early hyperstimulation syndrome (OHSS) - hyper-responder patients, Image 4.1.2.1]. Also it was found that multiple pregnancy rates in FET are very high and thus it is suggested to carry out single FET cycles. Alternatively, prior to a fresh ET cycle, the excessive ovarian hormonal exposure related to oocytes collection (not in FET) could entail a variety of negative effects on the very early pregnancy. A severe OHSS (poor-responder patients, Image 4.1.2.1) in cycles with fresh ET is possible to be prevented by using antagonist protocols with a GnRH agonist trigger or with embryo cryopreservation (Chang et al., 2017).

Image 4.1.2.1: Classification of Ovarian Hyperstimulation Syndrome (OHSS) (Abuzeid et al., 2014).

FET provides a better uterine environment (rather than the excessive hormone exposure in fresh cycles) for the embryos and improves the birth outcome. FET leads to lower rates of perinatal mortality and fewer birth defects. In fresh ET cycles there is risk of abnormal placentation and preeclampsia compared to the FET cycles (Weinerman & Mainigi, 2014; Zhang et al., 2018). In conclusion reports in favor of the frozen ET procedures are growing in number because of the improving IVF success percentages (Chang et al., 2017; Roque et al., 2013; Roque, Valle, Guimarães, Sampaio, & Geber, 2015).

4.2 Physiological and anatomical factors

4.2.1 Anatomy of cervix and uterus

The cervix is the lower part of the uterus in the human female reproductive system. The cervix is usually 2 to 3 cm long and its shape is cylindrical. The cervical canal runs along its entire length connecting the uterine cavity and the vagina. The opening into the uterus is called the internal os and the opening into the vagina is called the external os (Image 4.2.1.1). The cervix has a mucosal layer, a thick layer of smooth muscle, a covering of connective tissue connecting the cervical tube to the peritoneum of the abdomen. In front

of the upper part of the cervix lies the bladder that its filling is useful during ultrasonography in IVF cycles.

Image 4.2.1.1: The cervical canal (modified) (Thompson, 2017).

The uterus is the main hormone-responsive organ of the female reproductive system in humans and others mammals. It is responsible for the maintenance and transportation of the female gametes (oocytes). Within the uterus the fetus develops during pregnancy. The uterus is a thick-walled muscular organ capable of expansion to accommodate a growing fetus. From its lower part it is connected to the vagina through cervix and from its upper part to the fallopian tubes. It consists of 3 parts: the fundus (the top of the uterus, above the entry point of the uterine tubes), the body (site for implantation of the embryo) and the cervix (lower part of uterus linking it with the vagina) (Image 4.2.1.2).

Image 4.2.1.2: Anatomical parts of uterus and the cervical canal (modified) (Thompson, 2018).

By using the longitudinal axis of the body [Image 4.2.1.3 (a)] as the reference axis the position of the uterus can be described (uterine angles). The degree of the utero-cervical angle was divided into three anterior [anteversion (AV)] and three posterior [retroversion (RV)] positions depending on the size of the angle 1: 0O - 45O, 2: 45O - 90O, 3: > 90O resulting in six possible uterine positions [Image 4.2.1.3 (b)] (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017; Lesny, Killick, Tetlow, Manton, et al., 1999). Severe anteversion and

retroversion of the uterus has been identified as a factor of difficult transfer and cause diminished pregnancy rate (Garzo, 2006).

The passage through the cervical canal is usually described by three reference points: the internal, middle and external os of the cervix [Images 4.2.1.3 (b) and 4.2.1.4]. The passage through the cervical canal was considered as direct when the three reference points were aligned, and as not straightforward when the points were not aligned (Image 4.2.1.4) (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017).

(a)

(b)

Images 4.2.1.3 (a) and (b): Uterus and cervix anatomical features (b) (uterine positions are marked as AV: anteverted uterus and RV: retroverted uterus with each angle 1: 0 O - 45O, 2: 45O - 90O, 3: > 90O and for the cervical canal the reference as reference the longitudinal body axis (a) (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017).

Image 1.5.2.1.4: Cervical pathway with the external os (EO), middle os (MO) and internal os (IO) and the uterine angle (the common AV2 position). The external os is central, whereas the middle os is positioned slightly lower and off to the right. The internal os is also slightly to the right, horizontal to the external os (modified) (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017).

Cervical crypts are structures that are formed between the primary and secondary folds of the canal of cervix (Image 4.2.1.5). The depth and size of the crypts varies and the tip of the catheter may be trapped in one of them during transfer procedure. However crypts have not been reported as an important value which affects the success of transfer procedure (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017).

Image 1.5.2.1.5: A normal cervical canal, from the external to the internal os (a: cervical canal, b: primary and secondary folds, c: crypts and d: internal os) (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017).

4.2.2 Endometrial receptivity and endometriosis

Major abnormalities in the uterine cavity could be found in several women who are seeking treatment for infertility in ART programs. The most common pathological causes are

endometrial polyps, fibroids (also called leiomyomas or myomas) and chronic endometriosis (Image 4.2.2.1).

Image 4.2.2.1: Polyps, fibroids and endometriosis (modified) (Dr. Zelmanovich, 2016).

Uterine polyps are masses in the inner lining of the uterus due to abnormal growth of the tissue. They may also occur elsewhere in the body where mucous membranes exist like the cervix, vocal folds, and small intestine. Some polyps are tumors (neoplasms) and others are not neoplastic. In general the neoplastic polyps are benign, although some can be premalignant and/or malignant. Polyps could impact on fertility rates by disfiguring the endometrial cavity, having a detrimental effect on endometrial receptivity and increasing the risk of implantation failure. The impact of the polyp depends mainly on their number, size and location in the uterus (Check, Bostick-Smith, Choe, Amui, & Brasile, 2011; Rackow, Jorgensen, & Taylor, 2011; Stamatellos, Apostolides, Stamatopoulos, & Bontis, 2008; Yanaihara, Yorimitsu, Motoyama, Iwasaki, & Kawamura, 2008).

Uterine fibroids (also known as leiomyomas or myomas) are benign smooth muscle tumors of the uterus. Regarding their location the submucosal (they are located in the muscle beneath the endometrium of the uterus) and the intramural (they are located within the muscular wall of the uterus and are the most common type) fibroids distort and deform the endometrial cavity. They could be associated with poor pregnancy outcomes in women undergoing IVF treatment. Myomectomy should be considered as a method of treatment (Fatemi et al., 2010; Sunkara, Khairy, El-Toukhy, Khalaf, & Coomarasamy, 2010).

Endometriosis is a condition in which the endometrial tissue (the layer that normally covers the inner part of the uterus) grows outside of the uterus. Often the tissue can be found on the ovaries, fallopian tubes and uterus. The main symptoms are pelvic pain, dysmenorrhea and infertility. Some patients may be asymptomatic with only the symptom of infertility. The mechanism by which endometriosis causes infertility remains unclear. The distortion of uterine and peritoneal environment causes the accumulation of increased inflammatory cytokines and oxidative stress which may interfere with oocyte development, fertilization and embryo implantation and development (Miller et al., 2017).

Dysfunction of the immune system in endometriosis has become the main target of several studies. It is hypothesized that immunity plays a role in the pathogenesis of the disease. An increased number of B-cell is activated with the production of specific antibodies

against endometrial antigens, while T-cell and macrophage are not functioning properly (Ahn et al., 2015; Mier-Cabrera, Jiménez-Zamudio, García-Latorre, Cruz-Orozco, & Hernández-Guerrero, 2011; Miller et al., 2017). In addition, it has been observed that an increased production of cytokines and eicosanoids in the peritoneal area may affect sperm motility and velocity, penetration, embryo implantation, and early development (J. Wang, Shen, Huang, & Zhao, 2012). Furthermore it is not clear the role each abnormality actually plays in the pathogenesis of endometriosis-associated infertility.

The development of medical and surgical approaches, such as GnRH agonists protocols and laparoscopically-guided laser surgery have proven effective in improving many of the symptoms of endometriosis (Rossi & Prefumo, 2016). The transvaginal oocyte retrieval and the cryopreserved ET cycles have been associated with increased pregnancy success in endometriosis patients (Gomaa, Casper, Esfandiari, & Bentov, 2015; Queiroz Vaz et al., 2017; Surrey, Katz-Jaffe, Kondapalli, Gustofson, & Schoolcraft, 2017).

4.2.3 Cervical stiffness and stenosis

Cervical stiffness and stenosis may be congenital, iatrogenic, or secondary to infection, cervical trauma, endometriosis, or postmenopausal atrophy. The classic description of cervical stenosis is a narrowing of the cervical canal to less than 2.5 mm. Stenosis of the visible external cervical os may be visible. A transfer catheter cannot pass through the stenotic external cervical os or the external os cannot be clearly identified. The patient should return in the first days of the menstrual cycle since the menstrual blood may help identify the cervical os. In some cases, the patient may need a surgical intervention (dilation of the cervix) (Christianson, Barker, & Lindheim, 2008).

Stiffness and stenosis of cervix can usually be treated with mechanical dilation under anesthesia or conscious state. The patient presents to the operating room with a full bladder and placed in lithotomy position. A speculum is placed and the cervical external os is grasped with a tenaculum. The cervix is mechanically dilated under transabdominal ultrasound guidance. Also the vaginal tip and the cervix can be probed with a lacrimal dilator.

Cervical shaving (Hysteroscopic endocervical resection) could create a visible and compatible cervical canal. In this model a modified surgical hysteroscopic electrode is used to create a smooth cervical tract by shaving away approximately 0.5 millimetre (mm) of cervical tissue starting at the level of the internal cervical os and extending toward the external cervical os (Wortman & Daggett, 1996).

Another option to surgical intervention is the placement of cervical Laminaria sheets a few days prior to ET process. Laminaria are derived from compressed seaweed and expand in diameter as a result of the fluid they extract from the cervical canal lining. It has been shown that Laminaria can be used to successfully dilate the cervix prior to transfer (Glatstein, Pang, & McShane, 1997).

Alternative route in case of a difficult embryo transfer due to cervical stenosis and stiffness is the transmyometrial method where a higher pregnancy rate can be achieved as reported. The endometrial and myometrial trauma and contractions (in most cases of transcervical ET) are more excessive than in the short and non-invasive transmyometrial ET. The transmyometrial ET may bypass the hostile cervical environment and it is associated with less risk of catheter contamination and pelvic infections. Additionally the direct deposition of the embryos in the endometrium lining may ameliorate the implantation potential. Transmyometrial ET method could become an effective and safe procedure for patients (with certain anatomical abnormalities) who otherwise may have very difficult and traumatic transcervical ET. The only drawback is the risk of contractions of uterine junctional zone which becomes an important reason for pregnancy failure (Khairy, Shah, & Rajkhowa, 2016).

4.2.4 Psychological parameters and mental state

In many IVF clinics patients may ignore the risks of multiple pregnancies as most of them believe that “It won’t happen to me”. In fact, patients present with needs, desires, anxieties and stresses concerning their ability of bearing children. They are desperate and they are looking for a quick solution to their problem. Consequently, patients may not fully understand the dangers of multiple pregnancies and focus only on the potential benefit of having a child. It is valuable that ART physicians and psychotherapy specialists support their effort and help them to understand crucial steps of IVF procedures and risks (Klitzman, 2016).

Alternative treatment methods such as music therapy, herbal medicine, psychotherapy, massage and aromatherapy could reduce state and trait anxiety levels of patients who undergo IVF cycles. However the effect is not significant. In order to determine the success of the treatment, it is important to evaluate possible factors affecting the positive outcomes of the treatment and to conduct more studies with a large number of samples (Aba, Avci, Guzel, Ozcelik, & Gurtekin, 2017).

4.3 Technical approaches

4.3.1 Catheters and Syringes

4.3.1.1 Soft versus firm types of catheters

Several ET catheters are commercially available. All are composed of non-toxic plastics and vary in stiffness, malleability and physical attributes. Variations in ET catheter include stiff and soft materials, end and side openings and presence or absence of an outer sheath (Meriano, Weissman, Greenblatt, Ward, & Casper, 2000; Talwar, Naredi, Sandeep, Joneja, & Duggal, 2011). The most used soft catheters in ART science are the Wallace (Edwards-Wallace) and the Cook catheter while famous firm catheters are the Frydman, the

Tight Difficult Transfer (TDT), the Tomcat and the Rocket catheter (Images 4.3.1.1.1, 4.3.1.1.2, 4.3.1.1.3, 4.3.1.1.4 and 4.3.1.1.5). While stiff catheters and the use of an outer sheath make their placement easier, they may result in bleeding, trauma, mucus plugging and stimulation of uterine contractions. Soft catheters allow the tip to follow the contour of the cervical and uterine axis and minimize endometrial trauma. Nowadays variations of soft catheters are preferred by most IVF programs (Gardner et al., 2009).

Image 4.3.1.1.1: The soft Wallace catheter (Eugonia Assisted Reproduction Unit, n.d.).

Image 4.3.1.1.2: The soft Cook catheter (Talwar et al., 2011).

Image 4.3.1.1.3: The firm Frydman catheter (Talwar et al., 2011).

Image 4.3.1.1.4: The firm Tight Difficult Transfer (TDT) catheter (Monarch Medical Products, n.d.).

Image 4.3.1.1.5: The firm Tomcat catheter (DV Medical, n.d.).

Healthy and optimal quality embryos should be transferred in the most atraumatic method. The main goal of a successful ET is to smoothly traverse the cervical canal and the lower body part of the uterus with a catheter, to eject gently the embryos in the endometrial cavity and to avoid the retention of embryos in the catheter. An acceptable catheter for human ET procedure should be easy to use and should ensure proper placement in the uterus. Firm catheters with or without an outer sheath make their placement easier but may cause bleeding, mucous plugging, endometrial trauma and stimulation of uterine contractions. On the other hand, soft catheters allow the tip to follow the contour of the cervical and uterine axis and minimize the endometrial trauma (Ressler et al., 2013; Sigalos, Triantafyllidou, & Vlahos, 2017; Talwar et al., 2011).

Soft catheters provoke fewer traumas as they enter the uterine cavity causing less damage to the endometrium and may lead to improved clinical pregnancy rates. Despite the difficulty scale of an ET procedure, soft catheters are more flexible and pass more easily through the cervix and without the use of potentially traumatic instruments (tenaculum and obturator) (Murray, Healy, & Rombauts, 2003; Schoolcraft, 2016). This may be accomplished by using a priori trial embryo transfer by calculating the uterine cavity measurements and the utero-cervical angle prior to the actual transfer with the help of ultrasound means (Abou-Setta, 2006). Soft ET catheters (Wallace catheter and Cook catheter) may reduce the incidence of uterine contractions which may also affect implantation rates.

However, some scientists have concluded that when soft catheters pass through the cervical canal, in several cases ET could be difficult. In general ET is categorized as very easy when the catheter passed smoothly through the cervix. If the outer sheath is needed, ET is defined as moderate and if a tenaculum or an obturator is required, ET is difficult. Soft catheters are associated with a higher frequency of difficult transfers because they increase the use of a tenaculum or an obturator (Abou-Setta, Al-Inany, Mansour, Serour, & Aboulghar, 2005). Although the exact mechanism whereby these catheters improve clinical pregnancy rates is still unclear, soft catheters should be considered as the first-choice ET catheters (Buckett, 2006; Gardner et al., 2009).

Even though firm ET catheters are characterized with a low frequency of difficult transfers than softer catheters, they are not considered as adequate for ET use. A difficult ET procedure should not be considered as the only important factor in the pregnancy success potential (Abou-Setta, 2006). In this case the use of a tenaculum or an obturator could modify the utero-cervical angle and assist the cervical dilation that could not be overcomed by the catheter. These instruments can cause bleeding and uterine contractions which contribute to a decrease in ET success rates. Thus, these difficulties were resolved with the employment of a flexible soft catheter due to their ability to adapt to the cervical and uterine anatomy without the need of the aforementioned instruments and manipulations (Ruhlmann et al., 2015).

4.3.1.2 Echogenic catheters

Clinical pregnancy and embryo implantation rates were significantly improved with ultrasound-guided ET compared to clinical touch ET method. This has resulted to the production of ET catheters with echogenicity. Two different techniques are used to increase echogenicity of ET catheters. The most common manner involves the integration of a metal ring (< 2 millimeter, mm) close to the tip of the inner catheter. Examples of such echogenic catheters are the Cook Echotip and the Rocket EchoCat series. The metal ring is < 2 mm and only provides increased echogenicity toward the tip. On the other hand, the SureView catheters provide increased echogenicity through the entire length of the catheter (Buckett, 2006).

These catheters improve implantation and clinical pregnancy rates. Due to their visualization feature, they guide the catheter tip to the endometrial lining, minimize endometrial damage, correct the utero-cervical angle, avoid uterine contractions and deposit the embryos without touching the fundus (Ressler et al., 2013). Also echogenic catheters allow monitoring via abdominal ultrasonography with small movements, decreasing the events of difficult embryo transfers. It is of paramount importance to avoid difficult transfers. The time interval between catheter loading to embryos injection into the uterine cavity is a crucial factor of ET success. The use of echogenic catheters reduces procedure time by implementing an improved instrument in the ET and increases pregnancy rates. Thus, an easier transfer of embryos could be achieved.

The choice of echogenic catheters clearly facilitates the standardization of ET method, it simplifies ultrasound-guided ET and it minimizes the need for catheter movement to identify the tip into the uterus cavity (Urbina, Benjamin, Medina, & Lerner, 2015).

4.3.1.3 Types of syringes

Similar to the ET catheters, syringes should be sterile and non-toxic to the patients and the embryos. The syringes are used to create negative or positive pressure inside the catheter for the aspiration or release of the embryos. There are several types of syringe materials (Sigalos et al., 2017).  Norm-Ject plastic syringes (polypropylene and polyethylene material) (Image 4.3.1.3.1) are widely used and have a very small recoil effect due to the lack of latex at the end of the plunger.  Insulin syringes (polypropylene, rubber material and medical grade silicone oil lubricant) (Image 4.3.1.3.2) is used during ET process and their plunger should remain continuously compressed until the catheter is withdrawn from the uterus in order to avoid reaspiration of the embryos into the catheter due to recoil effect (Schoolcraft, Surrey, & Gardner, 2001).  Sterile glass syringes (glass material) (Image 4.3.1.3.3) can be used for embryo loading into the catheter.

Image 4.3.1.3.1: A Norm-Ject syringe (Fisher Scientific, n.d.).

Image 4.3.1.3.2: An insulin syringe (Birth International, n.d.).

Image 4.3.1.3.3: A sterile glass syringe (Science Company, n.d.).

There are three different kinds of syringes concerning the plunger: syringes with flat, conical and piston-like plunger (Images 4.3.1.3.4 and 4.3.1.3.5). The use of syringes with a flat plunger is more commonly used rather than syringes with conical and with piston-like plunger. Comparing syringes (flat, conical, and piston-like plungers) the type of syringe and the positioning of embryos within the catheter fluid column may have a significant effect on the speed of embryo release. Syringes with a conical or piston-like plunger provide less control of the release of embryos resulting in the abrupt propelling of embryos and damage of embryos caused by compression effect (Allahbadia & Chillik, 2015; Correa-Pérez & Fernández-Pelegrina, 2005).

Image 4.3.1.3.4: Syringes with flat and conical plunger (modified) (Mendat, 2016).

Image 4.3.1.3.5: Syringe with piston-like plunger (Cardinal Health, n.d.).

Whatever type of syringe is used in IVF laboratories, the specialist performing catheter loading and ET procedure should be familiar with all the physical attributes of syringes and catheters in order to avoid force and rough handling that could potential traumatize the patient.

4.3.2 Ultrasound guidance

4.3.2.1 2D ultrasonography

The development of ultrasound technology systems has improved medical and surgical precision. These image-guided visualization systems are using 2- or 3-dimensional (2D or 3D) magnetic resonance (Letterie, 2005).

Two-dimensional (2D) ultrasound has been reported to be an excellent real-time tool for tracking various surgical instruments and for monitoring a variety of endoscopic procedures. In reproductive medicine the position of the transfer catheter within the endometrial cavity may be monitored with this technique. However 2D ultrasound provides only limited views and low quality of images.

Under transabdominal ultrasonographic visualization, the cervix and the uterus can be visualized in 2 dimensions. The catheter tip is detected by 2D ultrasound imaging inside the endocervical canal and to the lower uterine segment. The embryo catheter is placed in an acceptable distance from the uterine fundus after passing through the cervical canal into the lower uterine segment. Then the embryos are transferred and the catheter is held in place for a few seconds. Then, it is gently removed from the patient always under ultrasound guidance (Allahbadia & Chillik, 2015).

4.3.2.2 3D ultrasonography

Three-dimensional (3D) ultrasound systems provide the opportunity to assess the 3 axis and to capture volume data. These volume data sets can be displayed by reconstructing images including 3 planes that cannot be obtained using conventional 2D ultrasound. This technique provides the possibility to view objects and it can optimize visualization of target issues. The ability to view the catheter location within the uterine cavity using 3 axis (plus volume plane) could provide an enhanced method of tracking the embryo catheter during ET process (Letterie, 2005).

During an ET process with a 3D ultrasound the cervix and the uterus can be visualized in 3 dimensions. During ET the catheter tip was monitored continuously during its journey to the cervical canal and to the lower uterine segment. A 3D ultrasound software constructs the images by implementing the volume data. The volume data are stored in a computer software for later recall and image reconstruction. With this system the images could be displayed at any desired orientation and at any location within the volume data set.

3D sonography offers a higher precision in catheter placement in the endometrial cavity. There was a significant increase in the clinical pregnancy and implantation rates between 2D and 3D ultrasound guidance in ET procedure (Cozzolino et al., 2018). Ultrasonography is a widely available tool in gynaecology and reproductive medicine and this technology may have a role in enhancing the efficiency of transfer techniques in IVF cycles as well.

4.3.2.3 Transvaginal versus abdominal method

Under ultrasound-guidance ET process, following the preparation of the cervix, the inner catheter is introduced into vaginal canal and is passed through the cervical canal into the uterine cavity. In the transvaginal ultrasound-guided ET the nurse holds the outer catheter while the physician guides the inner catheter to the correct endometrial location. In the abdominal ultrasound-guided ET the nurse holds the ultrasound transducer while the physician manipulates the inner and outer catheter set. The distance between the catheter tip and the fundus is measured again with ultrasonography. Embryos are deposited precisely in the middle third of the cavity. The catheter is removed gently, followed by the ultrasound probe. At the end of the procedure patients remain in bed for 20 minutes (Flisser, Grifo, Krey, & Noyes, 2006; Karavani, Ben-Meir, Shufaro, Hyman, & Revel, 2017; Larue, Keromnes, Massari, Roche, Moulin, et al., 2017).

The most common ultrasound technique in ART is the abdominal ultrasound while the transvaginal ultrasound is considered more effective. The proximity of the target and the transvaginal ultrasound transducer frequency allow an enhanced image quality compared to the abdominal ultrasound. With minimal or no trauma the embryo deposition in the uterine cavity is more precise concerning the height and width measurements (Cenksoy, Ficicioglu, Yesiladali, Akcin, & Kaspar, 2014). In several cases of difficult ET their main causes are tortuous cervical canals and cervical crypts. These abnormalities can easily be analyzed under transvaginal ultrasonographic guidance (unlike abdominal ultrasound which does not allow fine analysis of the cervix). This anatomical analysis enables to personalize the patient transfer by adapting the type of catheters and syringes, bladder filling and transfer bed position. Also the transvaginal ultrasound-guided ET enables the tip of the catheter to be visualized which can then be better guided into the cervical canal (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017; Larue, Keromnes, Massari, Roche, Moulin, et al., 2017).

However, there are speculations that ET would not be more effectively controlled during this technique. The manipulation of the catheter and the transvaginal transducer is probably considered technically difficult in a very narrow space into the cervical canal (Kojima, Nomiyama, Kumamoto, Matsumoto, & Iwasaka, 2001). Nevertheless, transvaginal ultrasound may allow a better management of transfer in IVF procedure due to precise embryo deposition and absence of mucosal trauma.

4.3.3 Types of embryo loading and volume (catheter wash and check - reload)

Embryos within a liquid medium may be loaded in an ET catheter for transfer into the uterine cavity with different loading techniques that may vary between clinics. Catheter- loading techniques may be essential for two types: (i) the air-fluid model in which the embryo with the transfer medium in the complex of syringe-catheter are separated with volumes of air spaces thus preventing the embryos from accidental spillage and (ii) the fluid- only method in which the catheter is entirely filled with the embryos and the transfer medium (whole-liquid phase) (Image 4.3.3.1) (Christianson et al., 2014; Moreno et al., 2004). A new loading method is reported as the “three-drop” procedure in which the embryos in transfer medium are separated by a bubble of air from a preceding and a following drop of medium (Tiras & Cenksoy, 2014).

Image 4.3.3.1: (1): Air-fluid technique, (2): Fluid-only technique (modified) (Moreno et al., 2004).

After grading and selecting the best embryos for transfer, it is time for embryo loading (same method is used on the two loading techniques):  The patient is headed and placed to the operating room into a lithotomy position.  The embryo culture dish with the respective embryos is removed from the incubator and embryos are loaded into the ET catheter.  A syringe is filled with culture medium (the syringe must not contain air bubbles).  The tip of the filled syringe is fixed to a catheter (it must be ensured that the fitting is tight otherwise the assembly may detach during critical point of ET process).  The entire medium is dispelled out of the catheter and the embryos are placed near the catheter tip.  The catheter is loaded with the embryos and once the syringe plunger is pushed, it is kept pressed until the withdrawal of the catheter (Releasing the pressure before complete withdrawal of the catheter could cause retained embryos due to negative pressure).  The catheter is slowly withdrawn after injecting the embryos.

 After the embryos have been transferred, the catheter is handed to the embryologist. The catheter is flushed by media and its tip is gently rolled on a lid and is examined for retained embryos, blood and mucus under the microscope.

The volume of embryos-transfer medium may have a significant impact on the implantation potential. High transfer volumes (> 60 microliter, μL) have been reported to result in expulsion of the embryos through the cervix into the vagina or a high risk of ectopic pregnancies (Nazari, Askari, Check, & O’Shaughnessy, 1993). However, the risk of an ectopic pregnancy depends on the position of the transfer catheter into the cervix and uterus (Montag, Kupka, van der Ven, & van der Ven, 2002). Thus, transferring volumes of < 30 μL is recommended to avoid these problems. The presence of air bubbles and smaller volume of ET medium with embryos (< 10 μL) are reported to present with a negative impact on the implantation and clinical pregnancy rates compared to higher volume (> 10 μL). Smaller volumes (< 10 μL) could result in additional air bubble formation negatively affecting implantation and pregnancy rates (Sigalos et al., 2017). Avoiding air bubbles results to a significant increase in implantation rate (Ebner et al., 2001; Eytan, Elad, & Jaffa, 2007a).

The air loaded into the transfer catheter in order to bracket the containing transfer medium with the embryos, has no negative effect on ET success (Krampl et al., 1995). Higher implantation and clinical pregnancy rates in the air-fluid technique are reported compared to the full fluid-only, despite the no significant difference in retained embryos. The employment of small air spaces in the catheter does not entirely affect the implantation and pregnancy rates provided that the total volume of transfer is small (10 μL) (Moreno et al., 2004). The air at the catheter tip is used to prevent spillage of the medium containing the embryos.The air-fluid loading technique enables embryos to retain their deposition place at the desired site in the uterine cavity (Abou-Setta, 2007).

4.3.4 Types of media employed for ET

Embryo transfer media are complex fluids containing various combinations of amino acids, carbohydrates, proteins and ions. The concentration of each component should be under control since changes in the composition may impact the embryo development and implantation (Biggers & Summers, 2008).

The most commonly used adherence compound into transfer media is hyaluronic acid (HA). HA is present in several female reproductive organs such as in the cervical mucus, the cumulus, the follicular fluid, the oviduct and the uterine fluids. The synthesis of HA is induced at the time of implantation (Nishihara & Morimoto, 2017). It is a glycosaminoglycan with a strong negative charge and it attracts a large volume of water. This hydration produces a high-viscosity solution which might facilitate embryo to be transferred and prohibit expulsion of the embryo (Gardner, Rodriegez-Martinez, & Lane, 1999). HA interacts with CD44 glycoprotein which is expressed on the embryos and on the endometrial lining (Campbell, Swann, Aplin, et al., 1995; Campbell, Swann, Seif, Kimber, & Aplin, 1995). Transfer medium enriched with HA increases birth rates compared to transfer medium with

no HA and transfer medium with lower doses of HA. The beneficial effect of HA on clinical pregnancy rates are only observed in women with poor prognostic traits such as multiple failed IVF cycles or advanced age (Bontekoe, Johnson, Blake, & Blake, 2014).

Another common medium substance for ET process is human serum albumin (HSA). Nowadays recombinant human albumin might replace the HSA as a protein source. HSA contains fatty acids in contrary to recombinant human albumin. The role of fatty acids is important in media of preimplantation embryos. Nevertheless high HSA concentrations in the medium fail to significantly improve ET success (J. Huang et al., 2016). Other proteins such as afamin have been reported to control and contribute more to embryo development in media (Nishihara & Morimoto, 2017). Studies employing the fibrin sealant as a compound in the ET medium concluded that there is no evidence of enhancing the outcome (Abou- Setta et al., 2014; Ben-Rafael et al., 1995; Bontekoe, Blake, Heineman, Williams, & Johnson, 2010).

In addition granulocyte-macrophage colony stimulation factor (GM-CSF) has been employed as a supplement to transfer medium (Siristatidis et al., 2013). GM-CSF is naturally detected in the female reproductive system and it can regulate embryo development by promoting glucose uptake and implantation. Thus it could increase embryo survival and live birth rates (Robertson, 2007; Ziebe et al., 2013). The beneficial effect of the GM-CSF can be attributed to its immunoregulative action on the endometrium.

Further future researches are required in order to clarify the effects of the substances of ET media on embryonic development and fertility success.

4.4 Overall Transfer procedure - performance

4.4.1 Number of embryos transferred

The most important reason for decreasing the number of transferred embryos is the need to decrease the high incidence of multiple gestation and multiple birth rates (MBRs) in ART. Despite that the majority of children born after multiple pregnancies are healthy, there are significant problems linked to multiple births both of obstetrical and neonatal nature (Gerris, 2005). The obstetrical risks include hypertension, preeclampsia, preterm labor, anemia, and increased cesarean section percentages. The neonatal risks include increased mortality, low gestational phase, low birth weight, neurological complications and respiratory distress syndrome.

Embryo reduction has been implemented to avoid the complications associated with multiple pregnancies. The only realistic way of avoiding multiple pregnancies in IVF is to reduce the number of embryos transferred. Thus, only one embryo should be transferred [Single Embryo Transfer (SET)]. Studies show a clear advantage of transferring two embryos compared to one embryo concerning implantation rates. Double Embryo Transfer (DET)

produces a significantly higher clinical pregnancy rate compared to Single Embryo Transfer (SET) (Baruffi et al., 2009). Based on patients’ data of ART programs, the following guidelines are recommended (Gatimel et al., 2017; Penzias, Bendikson, Butts, Coutifaris, Fossum, et al., 2017; Practice Committee of American Society for Reproductive Medicine & Practice Committee of Society for Assisted Reproductive Technology, 2013):  In patients of any age the transfer of one embryo is the most optimal option.  Regardless of the embryo developmental stage, patients under the age of 35 should receive a Single Embryo Transfer.  For patients between 35 and 37 years of age the Single Embryo Transfer is considered as the best option treatment.  For patients between 38 and 40 years of age three embryos in cleavage stage or two blastocysts should be transferred.  For patients 41–42 years of age four embryos in cleavage stage or three blastocysts should be transferred.

4.4.2 Uterus position

The perception of the uterine position [anteverted (AV) or retroverted (RV) and anteflexed or retroflexed due to fundus place, Table 4.4.2.1 and Images 4.2.1.3 (a) and (b) and 4.2.1.4] is valuable (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017). Prior to the actual ET, it is essential to prevent misdirecting the transfer catheter into the cervical canal into the cavity. So a few days prior to the real procedure a trial ET should be performed and the uterine position should be evaluated since the place of the uterus may change. In some studies RV uterus would often change location at actual ET and thus it is suggested that patients with a RV uterus at trial transfer should have a full bladder in order to later perform the procedure (Henne & Milki, 2004; Sallam, 2005; W.-J. Yang, Lee, Su, Lin, & Hwu, 2007).

Table 4.4.2.1: Description of uterine positions.

Description Most common position Less common position Uterus top Anteverted: Top is located forward Retroverted: Top is located backwards position Anteflexed: Fundus is placed forward Retroflexed: Fundus is placed backwards Fundus position relative to the cervix relative to the cervix

An ultrasound-guided trial transfer prior to the actual ET enables the estimation of the utero-cervical angle [angle categories: 1: 0O - 45O, 2: 45O - 90O, 3: > 90O, Image 4.2.1.3 (b)] (Larue, Keromnes, Massari, Roche, Bouret, et al., 2017; Lesny, Killick, Tetlow, Manton, et al., 1999). The angle correction allows direction of the catheter through the passage of cervical canal and avoids disruption of the endometrium, mucus plugging and bleeding. Also an accurate knowledge of the uterine position and utero-cervical angle enables the clinician to estimate the transfer difficulty and to ensure an atraumatic ET procedure (Sallam, Agameya, Rahman, Ezzeldin, & Sallam, 2002). This information increases clinical pregnancy and implantation rates and diminishes the incidence of difficult and bloody transfers.

Patients with large angles (> 60°) had significantly lower pregnancy rates. Performing ultrasound-guided ET with a full bladder is considered helpful in order to straighten the utero-cervical angle and improve pregnancy rates (W.-J. Yang et al., 2007).

4.4.3 Presence of air in catheter

Air can enter the uterine cavity either intentionally (from air buffers) or unintended during aspiration of the transfer liquid.

The use of air spaces in the transfer catheter has been proposed by some clinicians in order to identify the positioning of embryos in the catheter and uterus on ultrasound, to prevent transport of the embryos within the catheter and to protect the embryos from cervical mucus and from discharge before entering the endometrial cavity (Eytan, Zaretsky, Jaffa, & Elad, 2007; Krampl et al., 1995; Moreno et al., 2004). Clinical pregnancy rates appeared higher in cases where the air bubbles were closer to the fundus (a position of < 10 mm away from the fundal endometrial surface). An air bubbles injection led to more of the transferred liquid being carried towards the fundus by enhancing the potential for implantation (Eytan, Elad, et al., 2007a). However due to dispersion, the final position of the air bubbles cannot be predicted (Cenksoy et al., 2014).

Some scientists have suggested that the presence of air could be a non-physiological factor with a negative effect on the embryos and implantation. It could increase the likelihood of embryo entrapment, increase reactive oxygen species, the occurrence of retained embryos within the catheter, or chaotic dispersion patterns of the embryos released from the catheter (Correa-Pérez & Fernández-Pelegrina, 2005; Krampl et al., 1995). Air bubbles are formed during injection of liquid with air into the uterus, inhibiting the dispersion of the transferred liquid toward the fundus, spreading the fluid backward and causing its spillage out of the uterus. Embryos could be harmed directly. Hence the acceptable volume of air entering the uterus should be kept to a minimum in order to avoid embryo damage and to enhance the embryo chances of reaching the optimal site of implantation (Ebner et al., 2001; Eytan, Elad, & Jaffa, 2007b).

To conclude, the total volume of air (intended and unintended aspiration) should be kept to a minimum. Large volume of air is a limiting factor. Thus, by minimizing the entry of air into the catheter the quantity of embryo medium for transfer may be increased and the air contact with catheter tip should be avoided (Ebner et al., 2001).

4.4.4 Fluid dynamics and pressure

The pressure of medium with the embryos changes during the embryo loading process. By using the standard syringe-catheter complex it is easy to achieve a high pressure in the transferred embryos-medium volume and the pressure buildup in the transferred

liquid is related to the ejection speed of the syringe plunger. Furthermore in a short period of time pressure fluctuations are often observed (Groeneveld et al., 2012).

Cell damage can be caused by pressure fluctuations because of little attention paid to pressure changes during ET. The most common phenomenon is the presence of an air bubble in the medium liquid which causes a pressure gradient (Eytan, Elad, et al., 2007a). Although the embryos may then be found anywhere in the liquid volume, a greater probability of exposure and serious damage exists when it is localized in the front of the air bubble. It is a possible factor influencing embryo viability. There are four stages of pressure buildup during transfer procedure (Image 4.4.4.1) (Grygoruk et al., 2011). I. In the beginning of ET the transferred liquid leaves the catheter. II. Once the transfer liquid is delivered into the uterine cavity, the air bubble starts to grow. III. The growing air bubble exerts pressure on the transferred liquid which is surrounded by uterine fluid. It creates conditions for pressure buildup in transferred liquid. IV. In the end the air bubble is carried forward by pressure forces, the liquid concentrated in front of the air bubble is subsequently compressed to both sides and creates a local high-pressure buildup in the transferred liquid.

Image 4.4.4.1: Mechanism of the pressure buildup during ET (marks A, B, C, D and E represent A: uterine fluid, B: transferred medium C: catheter tip, D: air bubble and E: embryo as well as the short black arrows point the direction of the pressure forces acting on the liquid with embryo and the long black arrows show the movement direction of the air bubble) (Grygoruk et al., 2011).

The movement of the plunger during liquid injection causes a significant frontal acceleration of the transferred fluid. An increase in the injection speed provokes shear stress, dynamic pressure, and velocity difference. Furthermore a narrow catheter tip can increase the loading speed as well as the dynamic pressure. With high enough injection speeds stress can be strong enough to injure the vital cell’s organs and impair embryo viability (Groeneveld et al., 2012).

High pressure during ET can be achieved easily and thus it is crucial that ET process should be performed as slowly as possible to avoid injuring and stressing the embryos.

Moreover it would be optimal to maintain the embryos in the center of the catheter and far from the wall of the catheter because of minimal dynamic pressure and lower loading velocity.

4.4.5 Transfer depth of embryo placement

The cavity depth, estimated by ultrasound is clinically useful to determine the depth in which catheter insertion should occur. The clinical pregnancy rate is reported to be significantly affected by the transfer distance from the fundus. The rates of clinical pregnancy are increasing for every additional millimeter that embryos are deposited away from the fundus (Pope, Cook, Arny, Novak, & Grow, 2004; Y. Sun, Fang, Su, & Guo, 2009). Several studies have suggested the benefit of depositing embryos at a distance interval of 10-15 mm (millimeter) between the tip of the catheter and the uterine fundus at transfer (Pacchiarotti et al., 2007). An optimal distance of 10-20 mm seems to be the best site for embryo transfer to achieve higher pregnancy rates (Tiras, Polat, Korucuoglu, Zeyneloglu, & Yarali, 2010).

4.5 Observations during ET procedure and their importance (mucus, blood, comfort, reload, use of tenaculum / obturator)

4.5.1 Presence of mucus and blood in catheter

ET is the most crucial step for ART. Possible factors affecting implantation rates before, during and after ET have been widely researched (Schoolcraft et al., 2001). Blood and mucus on the catheter could affect implantation and clinical pregnancy rates (Tiras et al., 2012b).

Blood on the ET catheter may appear from two different sources. Cervical bleeding is probably the most common cause. This cervical bleeding may be the result of either traumatic passage of the catheter from the cervical canal or an infection such as bacterial contamination. Uterine bleeding is a sign of traumatic technical contact between the catheter and the uterine wall. Once trauma occurs, blood circulates in the endometrial cavity and alters embryo position. Blood in the endometrial cavity interferes with embryo implantation and uterine contractions resulting from trauma (Rhodes, McCoy, Higdon, & Boone, 2005; Sallam, Agameya, Rahman, Ezzeldin, & Sallam, 2003).

Cervical mucus on the catheter has a simpler explanation. Its appearance is associated with cervical contamination and mucus on the catheter is not expected to influence IVF success. A negative effect of mucus on the transfer catheter may be the phenomenon of retained embryos due to their blockage in the catheter (Rhodes et al., 2005). Thus mucus from the cervix should be removed prior to ET procedure in an effort to decrease the incidence of retained embryos (Craciunas, Tsampras, & Fitzgerald, 2014).

The presence of blood on the catheter on ET decreases implantation and clinical pregnancy rates. It is not always possible to determine the exact reason of bleeding. Regardless of the source it is clear that blood on the catheter is a marker for reduced success in ET. On the other hand, mucus on the catheter does not affect IVF success. Mucus on the catheter is reported to be a simple contamination with pregnancy rates remaining unaffected (Levi Setti et al., 2003; Moragianni et al., 2010; Spandorfer et al., 2003; Tiras et al., 2012b).

4.5.2 Difficulties during transfer process

A difficult ET significantly reduces the healthy pregnancy chances. Despite the constant advances of ART in safer and more efficient fertility protocols and laboratory techniques, better embryo culture media, valuable selection criteria, preimplantation genetic screening, and new simple cryopreservation method of vitrification for later IVF cycles, there have been no major changes in transfer procedure in recent years (Kava- Braverman et al., 2017).

The main ET purpose is to deposit gently and atraumatically the embryos inside the uterine cavity. It is crucial that this be performed in the correct bed position to achieve proper implantation (Schoolcraft, 2016). Different theories have been proposed to explain how a difficult ET may reduce pregnancy rates. Endometrial trauma and induction of uterine contractions influence correct embryo implantation. Increased age may be related to the difficulty of the ET (Phillips, Martins, Nastri, & Raine-Fenning, 2013).

ET was considered difficult when additional maneuvers and surgical instruments (use of outer catheter sheaths or tenaculum or obturator) are progressively applied in the procedure. Each additional maneuver, outer sheaths of catheter, tenaculum and obturator shows a progressive reduction in ongoing pregnancy and live birth rates (Levi Setti et al., 2003). Also poor ultrasound visualization (in the case of “clinical touch” transfer) can be crucial and clinical pregnancy rates are diminished significantly (Brown et al., 2016). Ultrasound guidance during ET is an important tool for improving the success transfer percentages. Thus a greater effort should be made to achieve a proper ultrasound assessment and an optimal visualization of the catheter during ET under is critical for the success of the procedure (Wood, Batzer, Go, Gutmann, & Corson, 2000).

Embryo expulsion, touching the fundus, injuring the endometrial lining and the presence of mucus in the catheter do not have a significant impact on implantation outcomes (Levi Setti et al., 2003; Moragianni et al., 2010; Tiras et al., 2012b) but they could be linked to the induction of uterine contractions the appearance of blood on the catheter diminishes the clinical pregnancy success (Phillips et al., 2013).

Specialists should realize that ET is essential to optimize ART outcomes and clinics should have an ET protocol which includes a description of the procedure and additional

maneuvers-techniques and instruments. It is important the knowledge of every additional applied movement that could lead to a reduction in the success rates (Levi Setti et al., 2003).

Considering the value of ET technique as the final step in ART, every effort should be made to optimize this process. An important classification for an ease transfer (Table 4.5.2.1) is applied and should be improved for ET future practices. This permits an evaluation of ET difficulty through certain techniques that would be applied in order and would lower the IVF success. Further studies could be designed and conducted in order to improve the transfer method (Kava-Braverman et al., 2017).

Table 4.5.2.1: Classification of ET difficulty.

Types of difficulty Description 1 Smooth completed ET without additional maneuvers and instruments 2 Embryo loading with an outer catheter sheath 3 Embryo loading with use of an obturator 4 Embryo loading with use of a tenaculum

4.5.3 Reload phenomenon

Embryo transfer is the last and the most important IVF stage. Even if it is performed very carefully, retained embryos in the transfer catheter may occur. Some of the risk factors associated with retained embryos at transfer may include (H.-C. Lee, Seifer, & Shelden, 2004; Schoolcraft et al., 2001):  the number of embryos transferred,  catheter contamination with mucus and/or blood,  technical difficulties during ET,  the physician experience performing the transfer,  the medium volume used during loading process,  catheter types, and  the developmental stage at the time of transfer.

The number of embryos transferred appeared to be the most important parameter. Reducing the number of embryos during ET process and transferring fewer embryos in order to prevent multiple pregnancies may also contribute to decrease the incidence of retained embryos (Vicdan et al., 2007).

Another common risk for retained embryos is the improper aspiration of cervical mucus prior to ET procedure. Embryos are not usually loaded when the catheter is contaminated with mucus and blood or when the transfer procedure is characterized as difficult. However the majority of embryos are successfully replaced at the second or third attempt. It is suggested that the removal of the cervical mucus before ET lowers embryo retention incidence and the immediate embryo retransfer is necessary (Craciunas et al., 2014; Rhodes et al., 2005).

Retained embryos and their immediate retransfer had no effect on clinical pregnancy and implantation rates. Nonetheless it is an unwanted event and every effort should be spent to avoid its occurrence.

4.5.4 Uterine contractions by use of tenaculum / obturator

The use of tenaculum and an obturator (Images 4.5.4.1 and 4.5.4.2) during ET process is still in doubt on whether it affects the pregnancy outcome. In IVF cycles this issue has been mentioned in many scientific studies. Tenaculum and obturator are used in many ART cases until nowadays. Their application to the cervix before ET can cause uterine contractions which may affect the pregnancy outcomes (Lesny, Killick, Tetlow, Robinson, & Maguiness, 1999; Schoolcraft et al., 2001; Strawn, Roesler, Rinke, & Aiman, 2000).

Image 4.5.4.1: Tenaculum (Mock Medical, n.d.).

Image 4.5.4.2: Catheter with a tip end and a mating obturator (“Presentation on theme: ‘Model 8709SC and Model 8731SC Intrathecal Catheters’ - SlidePlayer,” n.d.).

In cases of severely anteflexed and retroflexed uteri a tenaculum or an obturator on the cervix may potentially be required for traction at ET. Also their application may result in correct change of utero-cervical axis for an ease transfer. These surgical instruments are not generally recommended at the time of transfer because they may provoke uterine

contractions resulting in lower implantation and pregnancy success rates (Park et al., 2010; Rosenwaks & Wassarman, 2014).

The employment of tenaculum and/or obturator during ET may not affect the pregnancy outcomes due to other several risk factors influencing embryo viability. However more studies should be conducted in a larger population scale in order to conclude to concrete results (Chung et al., 2017).

4.5.5 Patient’s bed mobilization

The ET procedure is the last step of the IVF process and therefore is a very important procedure. Many factors have been proposed to increase the success of this stage. Various refinements of this technique have been suggested in order to improve the pregnancy and implantation rates.

To minimize the potential for movement and expulsion of embryos, different ART centers advise bed mobilization during ET. Bed rest is still advised despite the absence of scientific data for this practice. Comparing bed rest with no bed rest in hospital following embryo transfer has indicated that mobilization at the time of transfer does not reduce the chances of success (Sharif et al., 1998, 1995). Additionally bed mobilization has psychological implications for the patient. Since ART techniques are considered to be psychologically stressful, mobilization restrain patients from feeling comfortable and less nervous (Gallinelli et al., 2001; Rezábek, Koryntová, & Zivný, 2001).

There are inadequate evidences supporting the implementation of bed mobilization in hospital during transfer process to increase the implantation, clinical and ongoing pregnancy outcomes. Although resting in hospital is widely used and it is thought to be advantageous to the patient there is not enough clue that this practice could be beneficial.

5 Success rates following Embryo Transfer

5.1 Parental age

During IVF treatment procedures embryos might be negatively affected by paternal age. The implantation and ongoing pregnancy rates are decreasing especially in patients above the age of 35 (Quigley & Wolf, 1984).

The duration performance of transfers on advanced-age patients should be lower compared to the duration of the actual procedure because of the fact that embryos of women above the age of 35 would be more affected by environmental factors (such as light, oxygen levels, heat in the operating room). Many technological devices have been designed to protect oocytes and embryos from environmental damage during storage, manipulation and transfer. However it is considerably difficult to control all environmental factors during loading and discharging of embryos in the catheter. Therefore pregnancy rates decrease with longer time intervals spent in the catheter (Cetin, Kumtepe, Kiran, & Seydaoglu, 2010; Matorras, Mendoza, Expósito, & Rodriguez-Escudero, 2004). Although transfer durations has no effect on the clinical pregnancy rates of women below 35 years of age, clinical pregnancy rates of women above 35 years of age were significantly affected. The odds of no pregnancy achieved increases by 2 times in these women for longer-duration ET. These findings support the theory that gametes and embryos of women above the age of 35 are more sensitive to environmental factors than gametes and embryos of younger women (Kawahara, Ueda, Nakahori, & Honda, 2017).

In addition the possibility of finding fewer oocytes in patients aged above 35 is more advanced than in the patients below 35 years of age. Because of low numbers of oocytes the number of produced embryos is also lower. Thus, retrieved oocyte and transferred embryo numbers can be thought as factors increasing pregnancy failure in women above 35 years of age (Bdolah et al., 2015; Wen et al., 2013; Yan, Wu, Tang, Ding, & Chen, 2012).

5.2 Frozen versus fresh embryos

The main IVF strategy was always the transfer of one or more high-quality fresh embryos in the uterus. By the dawn of cryopreservation technology physicians are allowed to freeze any remaining embryos for subsequent use (Y. Shi et al., 2014). Although fresh ET still remains the primary treatment strategy, there is a growing view that elective frozen ET (FET) could result in better implantation and ongoing pregnancy outcomes.

The FET minimizes the risk of multiple pregnancies without decreasing implantation and birth rates (McLernon et al., 2010). Any supply of embryos that is available for future use, reduces the pressure and stress to transfer more than one embryo per cycle. This technique lowers the risks associated with twins or multiple pregnancies (Fauque et al., 2010; Gerris, 2009). Moreover during ovarian stimulation stage in a fresh IVF cycle super

high levels of hormones such as E2, P and other stress hormones may lead to uterine morphologic and biochemical changes and they might affect the endometrial receptivity and the embryo development. This situation could reduce the implantation rates (Devroey, Bourgain, Macklon, & Fauser, 2004). In ovarian stimulation phase of fresh IVF severe ovarian hyperstimulation syndrome (OHSS), a serious life-threatening complication could be occurred while frozen transfer method reduces the appearance of this condition (Nelson, 2017). When embryos are placed in women with severe OHSS, there is the danger of exposing them to a hostile uterine environment (due to high levels of estrogen) (Evans et al., 2014; Fatemi et al., 2014). This can be avoided by transfer in a subsequent natural or hormonal-depended cycle without affecting endometrial receptivity with the use of GnRH agonist. A more natural endometrial lining may be more beneficial for embryo implantation. Thus freezing of embryos with a view to replacement in a future natural or hormonally mediated cycle can reduce the risk of OHSS without decreasing pregnancy outcomes. Also pregnancies from frozen ET are associated with lower risks of obstetric and perinatal morbidity, antepartum hemorrhage, preterm birth and low birth weight (Maheshwari, Raja, & Bhattacharya, 2016).

The adoption of the policy of FET may rises concerns about its impact on the IVF success. Today birth rates from frozen ET cycles are comparable to those after fresh transfer process (Bhattacharya, 2016). However FET practice seems to be particularly successful in older women who undergo several ovarian stimulation processes and in women with very few eggs and embryos before attempting an ET of thawed embryos. Although FET perinatal risks appear to be low as possible, there are increasing odds of large gestational duration, higher mortality rates and offsprings born with body abnormalities after frozen ET cycles. This may be caused by embryo freezing and thawing (Wennerholm et al., 2013). To conclude nowadays FET is still a risky option for many clinicians and patients because they are aware that most of frozen embryos don’t survive at the freeze-thaw process.

5.3 Embryo developmental stage

Optimal-quality embryos have the ability to implant regardless of their zygotic score, time of early cleavage, cell number and fragmentation degree (0 - < 20%). Regarding female and male age, medical status and infertility factors, the number of top-quality embryos on days 2 and 3 is higher in patients with good clinical pregnancy outcomes (Sifer et al., 2011).

The female age has a negative impact on pregnancy success as well as the day of transfer and the number of good-quality embryos before transfer are associated with pregnancy success (Cetin et al., 2010). A decrease in pregnancy rates is found when ET is performed on day 2 compared to day 3. However top- and good-quality embryos obtained at days 2 and 3 has a significantly higher implantation success rate. Thus this feature would require excessively larger research studies for identification of its clinical value.

Several embryonic scores have been proposed to evaluate the number and the fragmentation degree of blastomeres (Terriou et al., 2007). It has been reported that 4-cell

embryos on day 2 have the best ability to implant and that pregnancy rates are reduced after transfer of embryos with unequal-sized blastomeres (Hardarson et al., 2001). Also an optimal cleavage pattern (from 4 cells on day 2 to 8 cells on day 3) has been associated with a high implantation potential (Magli et al., 2007). Day 3 embryos with 7–8 blastomeres were shown to have a lower aneuploidy rate compared with those with 5–6 or 9 blastomeres. Additionally it is not observed any difference between the ability to implant of embryos with fragmentation percentage from 0 to < 20% (Gerris et al., 1999).

Until today it has been proposed that the choice should be done according to the zygotic Z-scoring and the early cleavage grading system (Scott et al., 2000; Scott & Smith, 1998). Embryos with Z1 and Z2 scores have a higher ability to implant compared with Z3-Z4- scored zygotes. But the value of zygotic morphology in identifying embryos with high implantation potential is not obvious yet (Salumets et al., 2001). Also it has been showed that there is a decreased success rate when ET is performed on day 2 compared to day 3. It is likely to be related to the better selection of transferred embryo after a longer time period of culture (Blake, Farquhar, Johnson, & Proctor, 2007).

An immediate diagnosis of ectopic pregnancy is critical to avoid progression to tubal rupture and serious complications including hemorrhage and patient death. In order to decrease the risk of ectopic pregnancy scientists have been focused on the transfer technique, including embryo placement in lower uterus depth, avoidance of use of tenaculum at the time of ET, avoidance of the catheter touching the fundus and smaller volume of media injected during transfer (Lesny, Killick, Tetlow, Robinson, et al., 1999; Pope et al., 2004; Schoolcraft et al., 2001). Although these technical strategies have been applied in IVF by limiting the rate of an ectopic pregnancy, it is critical to consider the basic physiology of human reproduction in which the embryo in a natural conception does not enter the uterine cavity until the blastocyst stage (Bulletti & de Ziegler, 2006). Embryo transfer at the blastocyst stage on day 5-6 has been characterized as superior compared to day-3 transfer for having a higher implantation potential (Blake et al., 2007). Blastocysts have a larger diameter and a lower possibility of travel into the fallopian tubes thus decreasing ectopic implantation outcomes (Du et al., 2017; Smith, Oskowitz, Dodge, & Hacker, 2013).

In conclusion it is still unclear when an ET procedure during an IVF cycle should be performed preferentially. The predictive value of morphologic criteria requires further assessment in clinical trials with larger sample.

5.4 Loading technique

One of the important ET factors that could affect pregnancy rates is the embryo loading technique. Although the loading method is considered the most simple and easy step in the ET procedure, it could impact the ART outcome. Several loading techniques and equipment (catheters, syringes, volume of transfer media, medium concentration of protein,

catheter loading speed, medium viscosity and presence of air bubbles in the catheter) have been evaluated for their effect on success rate (Mains & Van Voorhis, 2010).

The most important issue of ET procedure is whether the catheter is firm or soft. ET catheters should be sufficiently soft to avoid endometrial trauma and malleable enough to be easily directed into the uterine cavity. Several studies have demonstrated that soft catheters have the best results in terms of pregnancy rates (Mansour & Aboulghar, 2002). Moreover by comparing of embryo loading directly from the culture micro drop versus loading from the transfer dish, no significant evidence has been reported in high successful pregnancy rates between these two methods (Halvaei, Khalili, Razi, Agha-Rahimi, & Nottola, 2013).

One key ET step during loading technique is the volume of the medium during embryo loading. The use of a small volume of medium (10–30 μL) has been proposed because of the fact that ectopic pregnancy may occur due to high fluid volume in the ET catheter. Moreover it was found that the transfer of a high volume of medium can increases the chance of dislocation of the transferred embryos from the uterus into the cervix and results in an ectopic pregnancy (Omidi, Halvaei, Mangoli, Khalili, & Razi, 2015). However it has been found that high fluid volume (> 30 μL) in the ET catheter increases the implantation and pregnancy rates compared to low fluid volume (15–20 μL) (Montag et al., 2002).

5.5 Presence of air bubble in uterus

The presence of an air bubble is a controversial subject. Some researchers concluded on the fact that there is no effect of air loaded into the catheter on the ET success rates (Moreno et al., 2004). In contrast, others demonstrated that air bubbles in the catheter with a small volume of medium (< 10 μL) had a negative effect on the implantation and pregnancy rates (Halvaei et al., 2013). However a new study has showed that the use of air brackets in air-fluid loading is neither beneficial nor detrimental when compared to the fluid-only method (Abou-Setta, 2007).

By comparing the two different catheter loading techniques (air-fluid and fluid-only approach) the presence of an air bubble in the catheter could be useful for tracking down the position of the embryos and the air bubbles during ultrasound-guided ET. The transferred air bubbles are often regarded as an indicator of embryo placement. Additionally the use of air bubbles around the embryo in the catheter might protect the embryos from the cervical mucus and accidental discharge before entering the endometrial cavity (Tiras et al., 2012a). However others believe that even a small amount of air in the uterus could be a non-physiological factor that has a deleterious effect on the embryos and implantation (Krampl et al., 1995). So a new loading method has been suggested in which only one air bubble is used at the tip of the ET catheter thus introducing less amount of air into the uterine cavity (Omidi et al., 2015).

Several studies analyzed the influence of the position of the catheter on pregnancy rates. For every additional millimeter of placement from the fundus the chances of clinical pregnancy may increase (Coroleu et al., 2002; Pope et al., 2004). Some studies have been shown that the majority of the transferred air bubbles show no movement after loading the embryos in the uterine cavity and a great number of good-quality embryos could implant successfully the area where they were initially transferred (Frankfurter, Silva, Mota, Trimarchi, & Keefe, 2003; Krampl et al., 1995).

The position of the embryos and the air bubbles at ET has been reported to influence pregnancy rates. Unfortunately during ET procedure it is not possible to predict and/or control their final position (Frankfurter et al., 2003). After loading the transfer medium the final position of the air bubbles (as well as of the embryos) depends on multiple factors such as the resistance of the syringe plunger, the pressure on the plunger and the withdrawal of catheter at the end of ET (Tiras et al., 2012a). Better implantation rates have been found when the transfer had been happened to a lower part of the uterus. Lower pregnancy odds have been found when the transfer had been happened towards the uterine fundus. The second ET method might have resulted in traumatic transfer and stimulation of frequent uterine contractions thus negatively affecting ET success. Therefore there is need for standardization of ET method that allows analyzing the value of exact position of embryo and air bubbles at transfer process (Lambers, Dogan, Lens, Schats, & Hompes, 2007).

5.6 Mobilization and bed rest after Embryo Transfer

Bed mobilization and rest is suggested to be performed for 20-30 minutes after ET by many IVF centers during a fertility treatment. It has been believed to achieve better pregnancy outcomes. On the other hand no evidence has been presented today to show that resting after transfer may negatively influence the ET success (Sharif et al., 1995).

Some ART clinics advise bed mobilization and rest for a longer time (from 1 hour up to 24 hours) with some restrictions in the patient’s daily life. Even though physicians suggest their patients to restrict some of their daily activities and to return to their normal routines several hours after ET, bed rest for a longer time is not associated with a better outcome of the IVF procedure (Lambers, Lambalk, Schats, & Hompes, 2009). In addition bed mobilization after ET has economic implications because of existence of free spaces in clinics, no extra costs for staying in hospital and immediate return to work (Purcell, Schembri, Telles, Fujimoto, & Cedars, 2007).

Women after ET are most vulnerable to psychological stress. The success of IVF treatment is depended on the levels of anxiety and depression. A great decrease in maternal stress with bed rest following ET might contribute to pregnancy rate (B. Li, Zhou, & Li, 2011; Yong, Martin, & Thong, 2000).

Nowadays there is currently not enough evidence to support bed rest after ET as being beneficial for women. Thus future studies and trials that investigate the effect of bed

mobilization and rest after ET should be performed and their results may be evaluated by women’s satisfaction for this practice, psychological factors and costs (Küçük, 2013).

5.7 Healthy or ectopic pregnancy

Most cases of ART treatments lead to successful delivery of healthy pregnancies (Kathpalia, Kapoor, & Sharma, 2016; Sha, Yin, Cheng, & Massey, 2018). However there are complications of pregnancy that some of them may develop more frequently:  Pregnancy loss: The cause of pregnancy loss may be implantation failure, fetal death or termination of pregnancy due to chromosomal and congenital abnormalities. Further loss reason is the incidence of multiple pregnancies (Pados et al., 2012).

 Risk of ectopic pregnancy: The cause of increased chances of ectopic pregnancy may be the migration of embryos or their direct transfer into fallopian tubes. Heterotopic pregnancies are extremely rare in women with natural conception but more common in infertile women who conceive under IVF treatment. In a heterotopic pregnancy there is one embryo which implants normally in the uterus and another which implants abnormally outside of the uterus (Du et al., 2017; Pados et al., 2012).

 Multiple pregnancies: Most of these pregnancies are twin pregnancies. The most common complication in these pregnancies is the low birth weight (Hack, Vereycken, Torrance, Koopman-Esseboom, & Derks, 2018; Vulid et al., 2013).

Women with IVF-conceived embryos are at increased risk of preeclampsia, gestational diabetes, placenta praevia and perinatal mortality. ART pregnancies also have higher relative risks of having induction of labor and cesarean section (Tarlatzis & Grimbizis, 1999). It has been reported that some of the children born to women who conceived with ART were premature and had respiratory difficulties (Henningsen et al., 2011). Nevertheless a long-term study in children conceived by IVF has demonstrated that the majority of children are developing normally (Olivennes et al., 2002).

6 Discussion

6.1 Latest trends in Embryo Transfer

Despite the increasing application of preimplantation genetic testing to ensure transferring euploid embryos as well as freeze-all policy to improve embryo-endometrium synchrony, the IVF success rates are still limiting until today. A simple technique may resolve this phenomenon and it is called endometrial scratching (ES) (also referred to as endometrial injury or trauma). It is a quick minimally invasive procedure with discomfort (Nastri, Ferriani, Raine-Fenning, & Martins, 2013; Santamaria, Katzorke, & Simón, 2016). It has been performed using a hysteroscope or a variety of soft and rigid endometrial biopsy devices (S. Y. Huang et al., 2011; Shokeir, Ebrahim, & El-Mogy, 2016) and catheters (such as a pipelle catheter) (Nastri et al., 2015) and involves causing intentional damage to the endometrium. Due to the fact that it is often performed in the preceding luteal phase, a disruption of an early pregnancy is the only underlying risk associated with this method (Goldberg, 2018).

Although the action mechanisms still remain unknown, it is believed that scratching improves the clinical pregnancy rates in women undergoing ART treatment (Wise, 2013). There are several hypotheses proposing a mechanism for improving endometrial receptivity with scratching. A successful implantation depends on factors involving several cytokines and growth factors because of the crosstalk between the embryo and endometrium. The main original hypothesis is that a local injury to the endometrium produced an inflammatory response resulting in a significantly increased number of macrophages, dendritic cells and cytokines [tumor necrosis factor-a (TNF-a), interleukins such as IL‑1, IL‑8 and IL-15 and macrophage inflammatory protein 1B (MIP-1B)] and thus increases the probability of embryo implantation (Gnainsky et al., 2010). In particular, high levels of TNF-a and MIP-1B have been detected during implantation suggesting the key role of immune system and inflammation in the development of a receptive endometrium (Haider & Knöfler, 2009). Cytokines stimulate the endometrial lining to attract monocytes and to induce their differentiation into dendritic cells which prepare the endometrium for embryo implantation (Gnainsky et al., 2015; Kalma et al., 2009).

With increasing evidence available it is important to reevaluate the effectiveness of endometrial trauma in different groups of women undergoing IVF. Endometrial scratching usually performed between day 7 of the previous cycle and day 7 of the transfer process and affects positively the IVF treatment by increasing clinical pregnancy, ongoing pregnancy and live birth rate (Nastri et al., 2015). However, it appears to improve the pregnancy outcomes only in women with more than two or more fail transfers by also taking into consideration the variables of the number and quality of embryos transferred and the timing of ES procedure (Nastri et al., 2012; Yeung et al., 2014). Evidence currently shows that it is not acceptable to perform endometrial injury routinely on all women undergoing IVF (Goldberg, 2018; Santamaria et al., 2016). This information is still considered to be unclear about the ES value (Mak et al., 2017). Some investigators have speculated that ES may have negative effects in pregnancy outcome (Aflatoonian, Baradaran Bagheri, & Hosseinisadat, 2016).

Thus, well designed trials with adequate sample are required in order to determine the purpose of endometrial trauma procedure in IVF cycles, not to cause uterine damage in the women as well as to get tangible results for women with repeatedly transfer or miscarriage failures.

Implantation is known to be a critical step for a successful pregnancy. It is a physiological interaction between the embryo and the endometrium (Bourgain & Devroey, 2007; Norwitz, Schust, & Fisher, 2001). Inappropriate implantation is the major cause in most IVF cycle failures (Simon & Laufer, 2012; Simur et al., 2009). Two main factors are believed to play a critical role in the implantation process: embryo quality and endometrial receptivity. Since the introduction of ART technologies several strategies have been developed to increase the implantation rate (Margalioth, Ben-Chetrit, Gal, & Eldar-Geva, 2006). In general, the success of implantation can be attributed to the action of multiple hormones and cytokines. Among these molecules involved in implantation human chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone which interacts with the endometrium (Makrigiannakis, Minas, Kalantaridou, Nikas, & Chrousos, 2006). hCG is required to maintain pregnancy and it is initially produced by the blastocyst 6–8 days after fertilization (Mostajeran, Godazandeh, Ahmadi, Movahedi, & Jabalamelian, 2017). This first embryonic signal initiates invasion and implantation of trophoblastic cells into the uterine myometrium and organizes the growth and differentiation of cytotrophoblasts to syncytiotrophoblasts. It also promotes the formation of villus trophoblast (Cole, 2010).

A normal implantation process requires the biosynthesis of multiple prostaglandins, proteinases and growth factors is a necessary step for a successful implantation. To stabilize an implanted embryo, adequate progesterone production, immune adjustment at implantation site, placental growth and differentiation are also needed (Norwitz et al., 2001). hCG is probably a critical signal in embryo stabilization. In particular it increases expression of the cyclooxygenase-2 gene resulting in increased production of prostaglandins (Zhou, Lei, & Rao, 1999). Additionally, hCG provokes the secretion of metalloproteinases in order to induce endometrial lining remodeling as well as increases IGF levels and angiogenesis (due to the increased intrauterine VEGF levels). hCG is an attractor of inflammatory cells such as neutrophils, monocytes and lymphocytes because this molecule inhibits macrophage phagocytosis activity and leukocyte factors in order to protect the embryo from the maternal immune system and to secure implantation. Also, it regulates endothelial cell responsiveness to IL‑1 and it has a cytokine-mediated effect on endothelial cell proliferation and migration. The mentioned above might be the possible mechanisms of hCG to improve implantation (Mostajeran et al., 2017).

During the past decades, various human trials have tested the hCG injection in ET. In a study, endometrial preparation was performed with progesterone and with an additional small amount of recombinant hCG on the day of ET and few days later (Ben-Meir et al., 2010). Implantation and pregnancy rates did not have significant correlation between the two-time space difference (14.9% implantation rate versus 12.7% pregnancy percentage and 32.2% versus 28.2% and p > 0.05). Most studies have concluded into the fact that administration of hCG at transfer has resulted in increased pregnancy rates. Intrauterine

injection of hCG has been associated with preimplantation changes of the endometrium (Banerjee & Fazleabas, 2010; Mostajeran et al., 2017). It has been shown that intrauterine injection of hCG before ET improves implantation and pregnancy rates (76.92% in patient group with hCG injection compared to 56.81% in patient group with no injection and 48.05% versus 33.33% and p < 0.05) (P. Huang, Wei, Li, & Qin, 2018). Another trial has demonstrated that the injection of hCG a few minutes after the intrauterine insemination procedure resulted in an improved pregnancy rate compared with 24-36h injection before the procedure (Järvelä, Tapanainen, & Martikainen, 2010). Moreover prior to ET the intrauterine injection of hCG has been performed in three different doses (100, 200 and 500 IU of hCG). The 500 IU of hCG improved significantly the implantation and ongoing pregnancy outcomes (Mansour et al., 2011). A recent augmentation in the implantation and pregnancy rates of women with intrauterine injection of 500 IU hCG has found to be beneficial in ET success (Aaleyasin et al., 2015). Although hCG injection has no negative effect on pregnancy outcomes, it could be associated with multiple gestations. Thus the injection of hCG may be more suitable in single ET methods (SET) where the risk of multiple gestations can be low enough and the implantation and pregnancy rates could be improved. Nonetheless further studies are needed to figure out its effect at the time of ET process. In conclusion an intrauterine 500 IU hCG injection at ET increases implantation and pregnancy rates. These findings suggest that hCG administration could be considered an adjuvant ET tool.

Synthetic compounds have been considered useful for transfer procedure for improving ET success outcomes. The prostaglandin synthetase inhibitors and oxytocin antagonists were suggested as uterine relaxants during the transfer procedure. Indomethacin as an inhibitor was studied in a trial in hopes of inhibiting prostaglandin release but there was no change in implantation rates (Bernabeu, Roca, Torres, & Ten, 2006). In addition oxytocin antagonists have been studied as a mean of improving ET outcomes. However it is not confirmed an improvement in implantation and pregnancy rates with their administration at the time of transfer (Ng et al., 2014). As previously it has been mentioned, one adjuvant reported to improve pregnancy rates is the intrauterine injection of hCG approximately 10 minutes before ET. In clinical trial a significant improvement in implantation and clinical pregnancy has been described with 500 IU of hCG injection at time of transfer (Mansour et al., 2011). Also ET medium has been linked to IVF outcome. Transfer medium with hyaluronan, a glycosaminoglycan found throughout the female reproductive tract, has been shown to improve pregnancy and live birth rates (Bontekoe et al., 2014). Recent studies concluded that it seems to be a benefit of hyaluronan in transfer media improving pregnancy rates (54.6% of patients with hyaluronan-enriched ET medium compared to 48.5% patients with transfer medium with absence of hyaluronan) (Urman, Yakin, Ata, Isiklar, & Balaban, 2008) (31.3% versus 4.0%, p < 0.0005) (Friedler et al., 2007) and implantation rates (32% versus 25%) (Urman et al., 2008). Adjuvant compounds should be suggested to be used during transfer procedure in order to achieve higher clinical pregnancy rates.

6.2 Optimal practice and the need for a universal protocol

Over the past years there has been a revolution of technologies in assisted reproduction (ART) that has allowed for progress in implantation rates. Refinements mainly in embryo selection criteria have allowed for the routine production of viable and good quality embryos “in vitro”. The evolving methods for embryo selection (time-lapse microscopy, preimplantation genetic diagnosis, proteomics and metabolomics) which are noninvasive techniques, seem to play a key role in the near future (Montag, Toth, & Strowitzki, 2013).

Despite these revolutionary improvements in the laboratory little has changed with the process of embryo transfer, the final step of IVF process. The technique of ET has remained relatively unchanged. ET has received little attention until recently. Nowadays numerous technical aspects of this method have been studied to minimize complications of this procedure and determine their effect on pregnancy rates and thus there is evidence that certain methods in the ET are associated with improved outcomes after IVF. Thus the current thesis will review various ET techniques performed during an IVF cycle, variables affecting ART success and approaches of optimization (Schoolcraft, 2016).

In general the procedure starts by placing a speculum in the vagina to visualize the cervix, which is cleansed with saline solution or culture media. Mucus in the cervical canal can be aspirated. A transfer catheter is loaded with the embryos and handed to the physician. The catheter is inserted through the cervical canal into the uterus where the embryos are deposited. The catheter is withdrawn and handed to the embryologist who examines it for retained embryos. The goal of a successful ET is to deliver the embryos atraumatically to a location in the uterus where the possibility of implantation is maximized (Mains & Van Voorhis, 2010).

Despite the simplicity of this procedure difficult transfers often occur and have been shown to significantly lower implantation and pregnancy rates compared with easy transfers (Wood et al., 2000). The difficulty of a transfer is often used to describe transfers that are time consuming, require a firmer catheter, cause discomfort or involve additional instruments such as a tenaculum and an obturator. Avoiding difficult ET performances is important to optimize clinical outcomes. First of all, the ultrasound guidance seems to be the main key in order to achieve this goal. An easy or intermediate transfer could result in a higher pregnancy outcome rather than a difficult one (p < 0.0001) (Tomás, Tikkinen, Tuomivaara, Tapanainen, & Martikainen, 2002). Also, a transfer procedure employing ultrasound guidance could be opted for as a mean of lowering the incidence of a difficult ET (odds ratio of 0.55), confirming catheter placement in the right part of the cavity and decreasing the chance of traumatizing the fundus as well as stimulating uterine contractions (Goudas et al., 1998; Sallam & Sadek, 2003). Compared with clinical touch ET, several studies have confirmed significant improvement in clinical pregnancy rates with ultrasound guidance (Abou-Setta et al., 2007; Brown et al., 2016). Transabdominal and transvaginal ultrasound seem to improve pregnancy outcome (Porat, Boehnlein, Schouweiler, Kang, & Lindheim, 2010). Additionally, several techniques including performing a transmyometrial

process and a trial transfer as well as the use of a soft catheter could be associated with easier ET. Despite the subjectivity of this matter an ease transfer is generally accepted as an important factor in successful implantation.

Ultrasonographic guidance is a crucial method used to facilitate atraumatic insertion of the catheter as well as to ensure correct embryo location of in the uterine cavity. Many physicians still use the clinical touch method without the help of ultrasound. The disadvantages of this method are the catheter contact with the fundus and the inaccurate assessment of the catheter position into the uterine cavity. This technique may lead to expulsion of the embryos and thus this phenomenon can be associated with lower pregnancy rates (Fanchin et al., 1998). Touching the fundus can easily be avoided with ultrasound. In one study based on transfers done by clinical touch and then visualized by ultrasound, the catheter tip contacted with the fundus in 17.4% of patients and with the fallopian tubes in 7.4% of cases (Woolcott & Stanger, 1997). In addition, in a recent review comparing ultrasound and clinical touch ET it has been concluded that ultrasound increases clinical pregnancy and birth rates (32.36% successful pregnancy cases, 26,14% live births and 1.64% ectopic pregnancies in the ultrasound-guided ET patients versus 26.91% pregnancy outcomes, 21,11% live births and 3,29% ectopic cases in the clinical touch ET patients, p < 0.0003) (Brown et al., 2016; Cozzolino et al., 2018). Furthermore, the visualization of the catheter at the utero-cervical angle can facilitate its insertion particularly in cases of severely flexed uteri. The full bladder is required for abdominal ultrasound and also helps to straighten the utero-cervical angle facilitating the catheter entry (Lewin et al., 1997; Lorusso et al., 2005). However, disadvantages with ultrasound may include a longer procedure time and the inconvenience of filling the patient’s bladder (de Camargo Martins et al., 2004). Also, in some studies ultrasound guidance has also been associated with fewer ectopic pregnancies (Sallam & Sadek, 2003; Tang, Ng, So, & Ho, 2001). Nowadays the echogenic catheters are available as ART laboratory equipment and help to identify the catheter location into the uterus. Some scientists have tried to use the method of 3D- or 4D- ultrasound concluding in encouraging early results (Gergely et al., 2005).

Trial or mock transfers can be done before the actual procedure. During the trial transfer a catheter is often placed to the fundus in order to measure the full length of cervical canal and uterine cavity. Any information and observations regarding the type of speculum, syringe and catheter, the need or not for a tenaculum or an obturator as well as the position of the catheter in the uterine cavity can be noted as references for the actual procedure. For example, during a trial transfer a stenotic cervix would not permit the passage of the catheter and thus a cervical dilatation can be planned before the actual ET. Techniques for cervical dilatation include the mechanical dilation (Prapas et al., 2004), the osmotic dilation with Laminaria sheets (Serhal, Ranieri, Khadum, & Wakim, 2003), the insertion of a Malecot catheter (Aust, Herod, & Gazvani, 2005) and the hysteroscopic shaving (Pabuccu et al., 2005). In most cases of cervical stenosis lower implantation outcomes are associated with cervical dilatation done within 5 days after the trial transfer but when dilatation is done several weeks before trial ET, it appears to improve pregnancy rates (Groutz, Lessing, Wolf, Yovel, et al., 1997; Prapas et al., 2004; Serhal et al., 2003). The longer time interval between the trial and the actual transfer allows the endometrium to

recover from any trauma, inflammation or bacterial contamination before the real ET process. Moreover some scientists have suggested performing a trial transfer at the time of oocyte retrieval. Although this could change endometrial receptivity a study has showed no difference in ongoing pregnancy outcomes when the trial was done before the start of ovulation induction compared with the one done at egg retrieval (47.6% versus 48.4%) (Katariya, Bates, Robinson, Arthur, & Propst, 2007). In conclusion the performance of a trial transfer was associated with a reduced incidence of difficult processes during the ET (29.8%). In a clinical study of trial transfer versus no trial transfer cases ongoing implantation and pregnancy rates were significantly improved in the trial transfer group (Mansour, Aboulghar, & Serour, 1990).

Transmyometrial ET is an alternative method to the transvaginal one and may be useful in patients with severe cervical stenosis or history of difficult transfers. Recently it was reported that pregnancy rates have not been increased with this technique over conventional transvaginal transfer (Groutz, Lessing, Wolf, Azem, et al., 1997). Due to increasing pain this method should be performed in only extremely difficult cases. Another method for such cases would be laparoscopic zygote intrafallopian tube (ZIFT) transfer.

In general, there are two catheter categories: soft and stiff. The ideal ET catheter is the soft ones (such as the Cook and the Wallace catheters). They tent to avoid trauma to the endometruim and they are malleable enough to be directed into the uterine cavity (Buckett, 2006). Their negative aspect is that in some cases of cervical stenosis they are more difficult to insert and sometimes require a malleable stylet or an additional instrument such as a tenaculum or an obturator. The use of those devices compared to the use of a soft catheter alone did not decrease clinical pregnancy or implantation rates (Abdelmassih et al., 2007; Silberstein et al., 2004). Also, a dilation of the cervix might be beneficial several weeks before ET and it was found to improve IVF outcomes (Serhal et al., 2003). On the other hand, firm catheters may facilitate placement in cases of difficult transfers but they can be associated with bleeding, trauma and stimulation of uterine contractions. Several studies have compared different types of transfer catheters. Soft catheters are preferred to firm ones (the Frydman, Tomcat and Rocket ET catheters) because they are less likely to induce cervical and endometrial injuries (McDonald & Norman, 2002; Sallam et al., 2003; Wood et al., 2000). In recent studies the use of soft catheters was associated with a higher pregnancy outcome than firm catheters (28,1% in soft versus 14,3% in firm catheters as well as 51,7% versus 27,6%) (Abou-Setta et al., 2005; Buckett, 2006). Ultrasound-guided transfers have demonstrated a disruption of the endometrium in 50% of patients when a Tomcat catheter was used compared to 12.5% with a Wallace catheter (Lavie, Margalioth, Geva-Eldar, & Ben- Chetrit, 1997).

A proper placement of the catheter tip is another important variable affecting ET outcome. The transfer distance from the fundus can be speculated as a variable affecting pregnancy rates (Pope et al., 2004). A transfer far from the uterine fundus may be more suitable. When the catheter was anywhere from 5 mm to 10 mm from the fundus, pregnancy rates were higher as well as ectopic pregnancy rates were lower. Depositing the embryos 15 mm from the fundus improved implantation compared with a 10 mm distance

from the fundus (60% in a 15 mm transfer distance compared to 39.3% in 10 mm) (Coroleu et al., 2002). Embryos deposited less than 5 mm from the fundus have a decreased implantation rate (Lesny, Killick, Tetlow, Robinson, & Maguiness, 1998; Pope et al., 2004; Woolcott & Stanger, 1997). Thus, pregnancy outcomes are increased with ET performed at the lower-middle uterine part (Coroleu et al., 2002; Franco et al., 2004; Frankfurter et al., 2003). Additionally, after a performance of an air-fluid technique the position of the air bubbles and its relation to pregnancy rates is also accessed. When the air bubbles were in the half part of the endometrial cavity, pregnancy rates were significantly higher (43%) compared with when the bubble was in the lower half part of the cavity (24.4%) (Lambers et al., 2007). More data have demonstrated that the air bubbles were located < 15 mm from the fundus pregnancy and implantation rates were significantly higher than those located > 15 mm from the fundus (Saravelos et al., 2016).

The speed of the embryo fluid injection could also be a hidden variable affecting ET outcome. In an interesting study a mock transfer was accomplished using a fast injection or slow injection speed of the embryos during the ET process (Grygoruk et al., 2012). In the rapid injection group, the embryos were shrunken and collapsed as well as rates of apoptosis were increased (wide range of apoptotic indexes: from 5% to 93% in the embryos exposed to high-speed injection ET). Also, embryo trauma and ectopic pregnancies could be observed. Thus, it was suggested that care should be taken while injecting the fluid as slowly as possible (Eytan, Elad, et al., 2007b; Eytan, Zaretsky, et al., 2007). Furthermore, pressure on the plunger of the syringe should be maintained until the catheter is completely withdrawn from the uterus in order to avoid a collision effect. The slow catheter withdrawal would minimize negative pressure. Some scientists suggest waiting before removing the catheter before the withdrawal. In a study a 30-second delay before catheter withdrawal did not significantly increase pregnancy rates (60.8% in patients with delay versus 69.4% in patients with no delay) (Martínez et al., 2001). In addition, a catheter column should be typically filled up with 20 mL fluid containing the embryos. Transfer volumes of more than 60 mL may result in expulsion of the embryos into the cervix. Volumes less than 10 mL may negatively affect implantation rates (Ebner et al., 2001). The embryos can be bracketed by air (air-fluid technique). The use of air bubbles in the catheter has slightly affected the pregnancy rates (36,5% in air-fluid method versus 30% in fluid-only method). The air bubbles are easily visualized on ultrasound and they can be a useful tool in transfer (Moreno et al., 2004).

Blood and mucus were associated with an increased risk for unsuccessful transfers. A study has demonstrated that a clinical pregnancy rate of 50% has been related with the absence of blood on the catheter tip and the other half rate has been correlated with the contamination of the catheter with a small amount of blood. When there was a significant amount of blood, pregnancy rates fell even further to 10% (Goudas et al., 1998). In another study, blood on the tip was associated with significantly lower pregnancy rates (42,1% in patients with absence of blood versus 29,3% in patients with severe blood amount) (Grady, Alavi, Vale, Khandwala, & McDonald, 2012). Cervical mucus can be responsible for plugging the tip of the catheter which may interfere with delivery of the embryos in the uterine cavity. Embryos may also be displaced in the mucus around the catheter tip or during

catheter withdrawal. So mucus can be aspirated with a sterile cotton ball or gauze soaked in saline or ET medium or with a sterile syringe attached to the catheter. The removal of cervical mucus was associated with increased pregnancy rates (49,5% in cases with no catheter contamination versus 44,44% in patients with presence of mucus in the catheter as well as 26,69% implantation rates versus 23,49%) (Eskandar et al., 2007).

An important aspect of ET is the presence of uterine contractions at the time of transfer. Monitoring contractions in patients undergoing ET has showed that when the number of contractions per minute increased, the pregnancy rates decreased (47,6% in ET with very low frequency of contractions versus 36% in ET with high frequency) (Fanchin et al., 1998). These contractions could be visualized by ultrasound guidance (Lesny et al., 1998). Contractions may be initiated by fundal contact or cervical manipulation associated with the release of prostaglandins (PGs) and oxytocin (Dorn et al., 1999). Recently oxytocin inhibitors have been infused to patients before ET in order to decrease the frequency of contractions at the time of ET and have demonstrated beneficial results in IVF success (Moraloglu, Tonguc, Var, Zeyrek, & Batioglu, 2010). Furthermore, embryos placed too high in the cavity may increase the probability of endometrial trauma and may induce uterine contractions (Abou-Setta et al., 2007). Middle cavity transfers seem to optimize implantation by avoiding the lower cavity where a suboptimal implantation could occur as well as some problems from traumatizing the endometrium at the fundus.

Another concern with ET is the possibility of expelled embryos. An analysis study has found that following ET embryos have been remained in the uterine cavity in only 58% of patient cases (Knutzen et al., 1992). After a routine transfer a 10% of patients had the embryos on the speculum where physicians rarely look for embryos in such locations (Poindexter et al., 1986). Embryos can also move back into the cervix whereby the catheter is removed. To minimize this phenomenon an additional amount of air after the embryo fluid catheter column was injected (air-fluid method) (Madani, Ashrafi, Jahangiri, Abadi, & Lankarani, 2010). This extra air injection resulted in a significant improvement in implantation and pregnancy rates. However, it should be noted that the presence of air bubbles does not completely alleviate this problem. During a recent study 12.4% of patients had air bubbles that migrated toward the cervix and their clinical pregnancy and implantation rates were significantly lower than in patients whose air bubbles and embryos remained static or moved toward the fundus (Saravelos et al., 2016).

The ET time is also an interesting variable involved in success. A study has demonstrated that the interval between loading the ET catheter and depositing the embryos in the uterus affected IVF outcomes. This time should be minimized because embryos may be vulnerable to exposure to the external temperature or light. A longer time interval has been shown to decrease ongoing pregnancy rates. When this delay was greater than 120 seconds, there was a decrease in pregnancy rates from 31.6% to 19.1% and a decrease in implantation rate from 15.9% to 9.4% (Matorras et al., 2004). The delay in injection might be a marker of the ET difficulty. Thus, minimizing the time between loading and transfer would be appropriate.

After the transfer the catheter should be handed to the embryologist to be examined for retained embryos. Any retained embryos should be properly reloaded for transfer into the uterus. One recent study reported lower pregnancy outcomes (3% versus 20.3%) when retained embryos were retransferred into the uterus (Visser, Fourie, & Kruger, 1993). Patients should be reassured that the phenomenon of retained embryos would be minimal and would have no effect on implantation rates.

Bed rest has been a controversial subject. Some physicians are recommending extended bed rest (24 hours) and some others are proposing no rest after the procedure. Recent studies have failed to demonstrate a clear improvement in ongoing pregnancy rates with bed rest (21,6% in ET with bed rest versus 21.3% in ET with no rest and 18% versus 22%) (Abou-Setta et al., 2014; Purcell et al., 2007). Some trials have even suggested a detrimental effect of bed rest on ET outcome (22% pregnancy results in patients with bed mobilization versus 50% in cases with no rest) (Gaikwad, Garrido, Cobo, Pellicer, & Remohi, 2013).

Frozen ET cycles have been indicated to significantly improve ongoing pregnancy and implantation outcomes compared to fresh transfers (Shapiro et al., 2011). There has also been evidence of improved obstetric outcomes in frozen transfers compared to the fresh ones (Holden et al., 2018; Roque, Valle, Sampaio, & Geber, 2018). Also, it was demonstrated that for frozen embryo transfers there was a lower incidence of preterm birth and low birth weight as well as a reduced need for intensive neonatal care (Pelkonen et al., 2010).

6.3 Future prospects

Over the past years there was a great progress and increasing pregnancy success rates in IVF. However, there was also present the risk of multiple pregnancies. While efforts are made in order to reduce the number of embryos transferred, the ongoing implantation and pregnancy outcomes are not ameliorating. Thus, the search for a marker of embryo quality for the optimal selection of a single embryo for transfer continues to be the major challenge. Selection methods based on morphological criteria and developmental dynamics. More recently several ART laboratories have utilized the preimplantation genetic testing for aneuploidy and they have concluded that that technique can be described as the best method to select the ideal embryo for transfer. Although this method predicts the best embryo selection and not a possible implantation rate (Harton et al., 2013). The most suitable technique should predictably choose the embryo that is most likely to implant and develop into a healthy baby (Rosenwaks, 2017). Nowadays there are several studies about novel non-invasive embryo assessment methods for evaluating gametes and embryos by quantifying mitochondrial DNA (mtDNA), microRNAs (miRs) and vitamin D as biomarkers for implantation potential.

Despite advancing techniques to identify good quality embryos prior to implantation, it is still difficult to determine why some embryos fail to implant. A recent

study has investigated whether there is a relationship between blastocyst mtDNA and morphologic traits (Klimczak et al., 2018). Embryos may differ from oocytes in that higher quantity of mtDNA is correlated with lower embryo quality. mtDNA is maternally inherited and mature oocytes contain all the mtDNA that will support the embryo development during the preimplantation stage. So, an embryo with higher amounts of mtDNA is more likely to be of poor quality. Similar studies exploring oocyte mtDNA have demonstrated that lower mtDNA content is associated with poorer quality oocytes and decreased ability to be fertilized (Chan et al., 2006; Desquiret-Dumas et al., 2017; Murakoshi et al., 2013; Santos, El Shourbagy, & St. John, 2006).

A comparison of mtDNA content in embryos that implanted versus those that did not, has indicated no statistically significant difference between these two groups. Thus, embryonal mtDNA may not be a good marker for implantation potential. Some trials are suggesting embryonal mtDNA content could be used to select embryos for transfer. Embryos with higher percentages (75% and 90%) of mtDNA content have appeared to be less likely to implant. This suggests that it may be more important to identify firstly the mtDNA content (Klimczak et al., 2018). Furthermore, it is known that implantation requires a significant increase in energy needs of the embryo and thus the utilization of mitochondria. Mitochondria react to stress by hyperproliferation and embryos that are under metabolic stress have a subsequent increase in mtDNA copy number (Monnot et al., 2013). It can be suggested that higher mtDNA content may be a marker of a stressed embryo. Also, when mutations are provoked into the mtDNA, faulty proteins and oxidative stress are produced in cells. It is possible that embryos under more oxidative stress have a particular mutation which is shared throughout most embryonic mitochondrial genomes. Moreover, these embryos are less likely to implant (Sobenin et al., 2014).

In an attempt to identify other potential markers for embryo viability, the expression profiles of some microRNAs molecules (known as miRNAs or miRs) were investigated in the chorionic villi of recently miscarriage women as well as in the peripheral blood of women undergoing the IVF-ET procedure (Q. Yang et al., 2018). The chorionic villi expression levels of miR-23a, miR-27a-3p, miR-29a-3p and miR-100-5p were significantly up in miscarriage patients compared to any other group. The expressions of these miRs were localized both in fetal trophoblast cells. miR-23a has been shown to promote trophoblast cell apoptosis and its expression level is up-regulated in the placenta (L. Li et al., 2017). Also, a significantly increased placental villi expression of miR-27a-3p might provoke a dysregulation of proliferation and the apoptosis of trophoblast cells resulting in pregnancy loss (Rah et al., 2017). The expression of miR-29a at the placental sites is evidently lower compared to other sites and its over-expression induces cell apoptosis (Gu et al., 2016). Thus, the significantly increased expression of miR-29a-3p in the placenta villi of miscarriage patients might be involved in the miscarriage pathogenesis by leading to excessive trophoblast cell apoptosis during early pregnancy. Also, the miR-100-5p expression was detected to be significantly elevated in these patients and its faulty expression might also induce trophoblast cell apoptosis resulting in embryo loss. Moreover, the decreasing villi expression of miR-127-3p in miscarriage patients suggested that it might be involved in miscarriage pathogenesis by reducing trophoblast cell invasion. miR-486-5p was another miRNA that showed also a

decreasing expression in this group of patients and its insufficient function might lead to implantation failure by the dysregulation of trophoblast cell activities (L. Shi, Liu, Zhao, & Shi, 2015).

Given that circulating miRs could reflect pathological condition and be detectable in peripheral blood, several miRs in the placenta of complicated pregnancies could be detected in maternal peripheral blood (Chen et al., 2008; Jiang et al., 2015). Compared to no pregnant women the plasma levels of miR-27a-3p, miR-29a-3p, miR-100-5p and miR-127-3p in miscarriage patients were significantly increased, whereas the miR-486-5p level was decreased. miR-27a-3p and miR-29a-3p could be potential non-invasive diagnostic biomarkers of pregnancy success during an IVF-ET cycle because increased levels of these miRs were observed both in the plasma and placenta of pregnant patients (Jairajpuri, Malalla, Mahmood, & Almawi, 2017; S. Yang, Li, Ge, Guo, & Chen, 2015). Moreover, an elevated serum miR-127-3p level was also demonstrated to be associated with small gestational age (Rodosthenous et al., 2017). Furthermore miR-29a-3p has been explored as a potential predictive biomarker for outcomes of the IVF process (Scalici et al., 2016). Thus, it has been observed an association between the peripheral blood levels of miRs and the outcomes of embryo transfer in an IVF procedure. The lower plasma miRs levels as well as the higher serum level of miR-27a-3p were observed to be correlated with the failure of ET. In particular the combination of plasma miRs levels reveals the outcome of IVF-ET with a sensitivity of 68.1% and a specificity of 54.1%. The combination of plasma miR-127-3p and miR-486-5p levels showed a slightly lower sensitivity (50.0%) but a notable higher specificity (75.3%) providing potential biomarkers to efficiently predict the outcomes of IVF-ET treatment (Q. Yang et al., 2018).

IVF science provides a unique opportunity to study the relationship of both systemic and intrafollicular levels of vitamin D with the oocyte fate, their follicular development, oocyte fertilization, embryo development, implantation and ongoing pregnancy outcome. Some systematic reviews of serum vitamin D and IVF outcomes do not support the serum screening of 25-(OH)-vitamin D in order to predict the clinical pregnancy rate because there were evidence suggesting that a lower serum vitamin D was associated with lower live birth rates (Lv, Wang, Wang, Wang, & Xu, 2016; Miklos, Li, Sorrell, Lyon, & Pielak, 2011; Pacis, Fortin, Zarek, Mumford, & Segars, 2015). In contrast present, findings have supported that 25-(OH)-vitamin D serum deficiency was associated with higher odds of live birth despite a higher miscarriage rate (Ciepiela, Dulęba, Kowaleczko, Chełstowski, & Kurzawa, 2018). Additionally, the relationship between vitamin D and oocyte quality may be influenced by the bioavailability of vitamin D within an individual follicle. Recent data have shown that 1,25-(OH)-2 vitamin D impacts in follicular survival and growth as well as oocyte growth (Xu, Hennebold, & Seifer, 2016). The high dose of 1,25-(OH)-2 vitamin D resulted to greater follicular diameter. However larger follicular size may not be linked to a higher quality oocyte.

Vitamin D supplementation during pregnancy is associated with increased circulating 25-(OH)-vitamin D levels, birth weight and birth length (Pérez-López et al., 2015). In contrast with this observation, previous studies have indicated that peripheral vitamin D status is a

reliable indicator of 25-(OH)-vitamin D availability within the ovary (Aleyasin et al., 2011; Anifandis et al., 2010; Firouzabadi, Rahmani, Rahsepar, & Firouzabadi, 2014; Ozkan et al., 2010). However, the mechanisms of action by which vitamin D may affect oocyte “health” still remains unclear. By evaluating vitamin D levels in follicles and monitoring them, local intrafollicular vitamin D levels strongly and negatively correlate with the oocyte quality (Ciepiela et al., 2018). Thus, intrafollicular concentration of vitamin D may be viewed as a marker of oocyte quality. Furthermore, the local actions of ovarian vitamin D (on pathways involved in the regulation of oocyte maturation) may contribute to oocyte ability to be successfully fertilized and developed into an embryo as well as it affects the possibility of pregnancy success in infertile patients undergoing IVF-ET treatments (Irani & Merhi, 2014; Merhi, Doswell, Krebs, & Cipolla, 2014; Wojtusik & Johnson, 2012). Nevertheless, previous studies examining 25-(OH)-vitamin D levels and IVF outcomes have demonstrated conflicting results. On the one hand a positive association between 25-(OH)-vitamin D levels and IVF outcomes in patients with the highest quantity of 25-(OH)-vitamin D could be more likely to achieve a good clinical pregnancy rate compared to patients with the lowest one (Firouzabadi et al., 2014; Ozkan et al., 2010). On the other hand, a negative relationship between increasing vitamin D levels and IVF rates has been demonstrated (Anifandis et al., 2010). Good quality embryos and a higher clinical pregnancy rate were found in patients with a vitamin D deficiency (Aleyasin et al., 2011). The lack of statistical significance (P = 0.17) could be related to the sample size and recent studies would need to include a relatively higher number of participants.

To conclude embryonal mtDNA has been suggested to hold an important role in predicting embryo viability. More trials are needed to examine the relationship between these markers and IVF outcomes. Further investigation of mtDNA both on clinical and molecular level is needed in order to understand how its content can be interpreted in order to increase pregnancy success in IVF treatment. In addition, the potential of circulating miRNAs as blood-based biomarkers for IVF treatment is promising. It has been suggested that peripheral blood levels of miRs were associated with the outcomes of transfer and they might present a diagnostic or predictive value for miscarriage. Moreover, the role of vitamin D is still unclear. Local concentrations of vitamin D may become useful to clinical decisions such as the selection of the best oocytes and thus may help to improve the results of IVF cycles. Further studies should evaluate how vitamin D may affect oocyte “health”. Such studies may provide an explanation for the relationship of vitamin D and IVF-ET clinical outcomes. The low abundance of these biomarkers, the inadequate sample sizes and insufficient utilization of available data should call for a large-scale multicenter trial in the near future.

6.4 Conclusions

Although major improvements in embryo culture, genetic screening, embryo selection and embryo cryopreservation have been described, little innovation has occurred regarding the ET method. The performance of an atraumatic ET is essential to IVF success. There are diverse studies which assess numerous parameters affecting the overall ET-IVF

result. Although every single factor has its impact on ET procedure, there are specific features which hold a crucial role in the transfer success. So these traits are the most studied and evaluated by many clinical trials over the years of ART science and research.

Ultrasound means is the most important and favorable factor influencing the transfer operation by increasing clinical ongoing pregnancy rates and reducing ectopic occasions (32.36% successful pregnancies, 26,14% live births and 1.64% ectopic cases in the ultrasound-guided ET patients versus 26.91%, 21,11% and 3,29% in clinical touch ET patients by Brown et al., 2016; Cozzolino et al., 2018). Another major aspect is the use of soft catheters. They were associated with positive outcomes of IVF treatment (28,1% in soft versus 14,3% in firm catheters, 51,7% versus 27,6% and endometrial injury in 12,5% of patients with soft catheters versus 50% with firm ones by Abou-Setta et al., 2005; Buckett, 2006; Lavie et al., 1997). Moreover the constant of blood and mucus is linked with unsuccessful IVFs. Blood presence could result in lower pregnancy chances (50% in patients with absence of blood versus 50% in cases with blood contamination and 42,1% versus 29,3% by Goudas et al., 1998; Grady et al., 2012) and reduce pregnancy rates further to 10% (Goudas et al., 1998). Mucus can be responsible for blocking the catheter which may interfere with the embryo delivery in the uterus. Thus the removal of mucus was slightly associated with positive pregnancy outcomes (49,5% in cases with no catheter contamination versus 44,44% in patients with presence of mucus in the catheter and 26,69% implantation rates versus 23,49% by Eskandar et al., 2007). Nonetheless the most important key fact in the ET success is the observation of the total sum of variables and their consequences during the overall process. Thus more clinical studies (RCTs) should be conducted in the near future for providing valuable information (mainly understanding the weighting factors affecting ET method) in order to contribute to ET-IVF technique outcome and to achieve a successful treatment.

To sum up based on various studies, trials and guidelines, there are some crucial main steps and details of the transfer process that clinicians should pay attention to. Experts recommend that trial transfers should be performed in order to avoid difficult transfers and allow better preparation for difficult cases. Ultrasonographic guidance is a very useful tool to ease transfers and result in improved pregnancy and live-birth outcomes. Furthermore, soft catheters should be employed during the ET process as well as cervical mucus should be removed in order to decrease plugging of the catheter. Embryos should be deposited in the middle part of the uterus. Following transfer, negative pressure should be minimized during the catheter withdrawal by maintaining the plunger of the syringe constantly pressured. The overall ET procedure should be performed in the minimum amount of time. Several future studies should be conducted in order to examine and evaluate other aspects of ET procedure. ET technical trends namely endometrial scratching, hCG infusion, adjutant compounds and biomarkers should be further investigated to assess their strength and potential to become valuable tools in order to achieve the maximum success rate of IVF-ET treatment, a healthy ongoing pregnancy and finally a healthy offspring.

Summary

In vitro fertilization (IVF) is a procedure in which fertilization of an oocyte is performed “in vitro” in a laboratory. The fertilized oocyte (zygote) is cultured under strict protocols and supported through its developmental journey to the preimplantation stages. The high quality embryos are being selected for transfer to the woman's uterus in order to achieve a healthy clinical pregnancy. Over the past years there has been a revolution of technologies in assisted reproduction (ART) that has allowed for progress in implantation and ongoing clinical pregnancy rates. This evolution has provided the physicians and embryologists the opportunity to use advanced embryo selection criteria, embryo culture and transfer techniques in their routine practice. Improvements mainly in selection criteria (time-lapse microscopy, preimplantation genetic diagnosis, proteomics and metabolomics) have allowed for the routine production of viable and good quality embryos.

Embryo transfer (ET) is the final and critical step in the success of in vitro fertilization, where embryos of either day two, day three or day five are transferred in the uterine cavity through the cervical canal. The aim of ET is to gently place the embryos in the endometrial cavity. Despite these revolutionary refinements in the laboratory little has changed with this process of embryo transfer. From the spring of IVF industry ET has received little attention until recently. Nowadays numerous technical aspects of this method have been studied to minimize complications of this procedure and determine their effect on pregnancy rates. Thus, various ET variables might impact pregnancy rates after an IVF cycle.

In general, the transfer procedure starts by placing a speculum in the vagina to visualize the cervix. Mucus in the cervical canal can be aspirated by cleansing the cervix with saline solution or culture media. A transfer catheter is loaded with the embryos and handed to the physician. The catheter is inserted through the cervical canal into the uterus where the embryos are deposited. The catheter is withdrawn and handed to the embryologist who examines it for retained embryos. The goal of a successful ET is to deliver the embryos atraumatically to a location in the uterus where the possibility of implantation is maximized.

Despite the simplicity of this procedure difficult transfers often occur and have been shown to significantly lower implantation and pregnancy rates compared with smooth transfers. These uneasy transfers might be a result of multiple technical aspects and factors affecting the success of embryo transfer. A notable example might be the endometrial receptiveness and other medical issues considering the endometrial quality are critical conditions affecting the ET outcome. Several studies have attributed infertility and failed IVF cycles to endometriosis and to various crucial factors with respect to the endometrial quality. Identifying a hostile environment for implantation as well as ways to manage it are important factors regarding ET success.

The embryo selection is mostly based on morphological criteria considering the developmental stage and embryo’s morphological characteristics. Important traits that are

evaluated daily during embryo culture are the zygote quality (pronuclear scoring system), the cleavage rate, as well as the number, size, symmetry and fragmentation of blastomeres. These features have been reported to present with high predictive value regarding pregnancy rate and implantation potential. Numerous evaluating scenarios and their validity have been reported however the lack of an optimal universal protocol of evaluation still stands. Furthermore, the quality of embryo includes the aspect of their fresh or frozen state. Frozen embryos (FETs) significantly achieve higher ongoing pregnancy outcomes compared to fresh embryos (fresh ETs). There has also been evidence of improved obstetric outcomes in frozen transfers compared to the fresh ones. Also, it is supported that for frozen embryos there was a lower incidence of preterm birth and low birth.

An important ET technical concern is the type of the transfer catheters regarding their design, quality of materials (stiff or soft), end and side openings, presence of an outer sheath and flexibility. Stiff catheters and presence of an outer sheath have been reported as useful tools for promoting embryo transfer. However, they are linked to cervical and endometrial bleeding or mucus plugging, phenomena that must be avoided for safety reasons regarding embryos and patients. On the other side soft catheters present more advantages as they follow naturally the cervical and the axis of uterine orifice minimizing trauma. Nowadays these catheters are preferred by most IVF programs due to the smoother ET they provide and the higher chance they present for clinical pregnancy. Additionally, syringes are employed in order to create negative or positive pressure inside the catheter and help release of embryos in the uterine cavity. There are different types regarding their shape (flat, conical, piston-like plunger) and material (plastic or glass). Conical or piston-like plunger syringes present with less control during the release of embryos. ET must be conducted gently to secure embryos’ protection and integrity.

Ultrasound-guided ET helps the transfer of embryos into the uterine cavity by facilitating an atraumatic insertion of the catheter as well as ensuring a correct embryo location of in the uterine cavity. Many physicians still use the clinical touch method without the help of ultrasound. The disadvantages of this method are the catheter contact with the fundus and the inaccurate assessment of the catheter position into the uterine cavity. This technique may lead to expulsion of the embryos and thus this phenomenon can be associated with lower pregnancy rates. Touching the fundus can easily be avoided with ultrasound. The ultrasound assists the evaluation of the depth while performing ET into the uterine cavity and providing the ability to visually record the ET process. The ultrasonographic technology ensures an accurate delivery of embryos to the site of implantation. Nowadays the echogenic catheters are available and help to identify their location into the uterus. Some scientists have tried to use the method of 3D- or 4D- ultrasound concluding in encouraging outcomes.

the position of the catheter in the uterine cavity can be noted as references for the actual procedure resulting in higher chances of a successful actual ET procedure.

Blood and mucus were associated with an increased risk for unsuccessful transfers. Blood on the tip is usually associated with significantly lower pregnancy rates. Cervical mucus can be responsible for plugging the tip of the catheter which may interfere with delivery of the embryos in the uterine cavity. Embryos may also be displaced in the mucus around the catheter tip or during catheter withdrawal. So, mucus can be aspirated and its removal is linked to increased ongoing pregnancy rates. Another crucial aspect of ET is the presence of uterine contractions at the time of transfer. Contractions are initiated by the release of prostaglandins (PGs). Embryos placed too high or too low in the cavity may increase the probability of endometrial trauma and may induce uterine contractions. Thus, middle cavity transfers seem to optimize implantation. The phenomenon of expelled embryos is also a grave concern. Embryos can also move back into the cervix when the catheter is withdrawn. To minimize this phenomenon an additional amount of air after the embryo fluid catheter column is often injected (air-fluid method). This extra air injection results in a significant improvement in clinical pregnancy outcomes.

After the transfer the catheter should be handed to the embryologist to be examined for retained embryos. Any retained embryos should be properly reloaded for transfer into the uterus. Also bed rest has been a controversial subject. Some physicians are recommending extended rest and some others are proposing no rest after ET.

To accomplish high success rates of ET, there are certain techniques claiming to assist the embryo implantation. The endometrial scratching (also known as endometrial trauma or injury) is a procedure where the endometrial lining is purposely traumatizes with special surgical instruments such as biopsy devices or a pipelle catheter. This method could cause some discomfort or pain. It is thought that this disruption may increase the chance of an embryo implantation and an ongoing pregnancy. Hormones and immune response are released to help the uterine lining repair itself. Therefore, this temporary injury seems to make the endometrium more receptive to an embryo. Moreover before ET an injection of a small quantity of human chorionic gonadotropin (hCG) (a hormone produced by the placenta after implantation) in patient’s uterus has shown beneficial results in the embryo implanting. The hCG assists the endometrial receptivity for embryos. This technique could assist in increased percentages of successful pregnancies. In addition, there is growing evidence and attention paid in developing a suitable technique which should predictably choose the embryo that is most likely to implant and develop into a healthy baby. Nowadays there are several studies about novel non-invasive embryo assessment methods for evaluating gametes and embryos by quantifying mitochondrial DNA (mtDNA), microRNAs (miRs) and vitamin D as biomarkers for implantation potential.

Several variables associated with ET have been studied in an effort to increase the efficiency in the success of the IVF process. Although the subject of “how to ensure an optimal ET” still remains the topic of interest, it has become evident that literature lacks reports on an overall approach of the process. Various aspects of ET procedure should be

studied and evaluated in future investigations. Thus, they might be considered as valuable details to take into consideration in order to achieve a successful IVF “journey” until ultimately delivering a healthy offspring.

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