**2. Endometriosis and embryo quality**

Among investigators who reported poorer results of IVF-ET outcome in patients with endometriosis, there is a general agreement on few final consequences in contrast to numerous possible pathophysiologic mechanisms leading to it.

These include:


An indirect marker of oocyte quality and a possible predictor of embryo's implantation capacity is the quality of the developing embryo. As such, this parameter could be used for assessment of the effects of endometriosis on fertility.

#### **2.1 Measures of embryo quality**

384 Endometriosis - Basic Concepts and Current Research Trends

In severe cases of endometriosis, the seriously distorted anatomy of pelvic organs is the obvious cause of impaired fertility. In the absence of pelvic adhesions and scarring, when only mild to moderate endometriotic lesions are present, finding the cause and the consequence is anything but an easy task. Many confounding factors make these studies both controversial and difficult to interpret. One of the main problems is the choice of the appropriate control group. The common practice has been to choose women with tubal factor infertility or those with unexplained infertility as controls. The problem with this choice is our inability to identify women with inherently reduced potential for conception (fertilization and implantation) and to exclude them from the control group. Also, the common practice is to make observations on the patients with endometriosis who are treated by one of the techniques of assisted reproductive technologies (ART). This practice is problematic because the use of ART creates a non-physiologic environment and many subtle but important *in vivo* effects of endometriosis on the process of conception may be hidden in

Hypotheses on mechanisms by which mild to moderate endometriosis could impair fertility potential are numerous and will be mentioned here only briefly. One group of investigators tested hypotheses that endocrine abnormalities in women with mild and moderate endometriosis might be the cause of reduced fertility. As was already conveniently summarized in the literature (Garrido et al., 2000; Hunter et al., 2004), proposed mechanisms were hypersecretion of prolactin in patients with endometriosis (Muse et al., 1982), impaired folliculogenesis (Tummon et al., 1988), altered ovulation (Dmowski et al., 1986) and luteal phase defects (Grant, 1966). The other line of investigation was directed towards immune dysfunctions as potential causes of infertility in patients with endometriosis. Proposed mechanisms were chronic inflammatory reaction and altered immune responses induced by endometriosis (Harada et al., 2001), including increased production of cytokines and other soluble immunomodulators in the peritoneal fluid. These altered immune responses could further affect motility and velocity of the sperm, lead to sperm phagocytosis (Soldati et al., 1989), accelerate ovum transport (Croxato et al., 1978), impair the process of fertilization (Mahadevan et al., 1983), display direct embryo toxicity (Damewood et al., 1990) or adversely affect the process of implantation (Yovich et al., 1985; Matson & Yovich, 1986). Unfortunately, results of these studies were usually contradicting and no definitive conclusion could be made so far. It is likely that there is more than one answer to this

Among investigators who reported poorer results of IVF-ET outcome in patients with endometriosis, there is a general agreement on few final consequences in contrast to

An indirect marker of oocyte quality and a possible predictor of embryo's implantation capacity is the quality of the developing embryo. As such, this parameter could be used for

1. Impairment of the quality of oocytes (resulting in lower fertilization rates) and/or

2. Decrease in the implantation capacity of embryos (Pellicer et al., 2001).

*in vitro* conditions.

complex problem.

These include:

**2. Endometriosis and embryo quality** 

numerous possible pathophysiologic mechanisms leading to it.

assessment of the effects of endometriosis on fertility.

Quality of the embryo may be described using many direct and indirect measures. For example, parameters which could be used for indirect assessment of embryo quality are number of embryos on Day 2, total number of blastocysts, number of frozen blastocysts, but also the implantation rate and early pregnancy loss rate. The only direct measure of embryo quality is embryo quality score based on morphological characteristics of a developing embryo. However, this parameter is quite difficult to use in practice. One reason for this difficulty is the absence of uniformity of scoring systems used by different laboratories. Another is a consequence of two facts: (1) quality score is a categorical variable and (2) it is (still) a common practice to transfer more than one embryo (blastocyst) at a time. In other words, if two or more embryos of different qualities have been transferred, it is not possible to calculate "the mean embryo quality score" or to determine exactly which one of transferred embryos has eventually implanted.

Researchers who would come across this issue tried to overcome it in various ways. The simplest was to exclude from the study those patients in whom embryos with different quality scores were transferred (La Sala et al., 2005). The main drawback of this approach was a significant reduction of observed cycles, i.e. of the sample size. Also, this tactic prevented incorporation of some other important variables (namely the number of transferred embryos) in the final analysis. On the other hand, some of the authors (Lambers et al, 2007) used a cumulative embryo score, previously introduced by Steer (Steer et al., 1992). Cumulative embryo score was defined as an additive parameter (i.e. following the transfer of two embryos with scores of 1 and 3, the total score of embryos transferred was 4). Other investigators (Winter et al., 2002) assessed embryo quality with relation to the number of embryos transferred and the possibility of elective transfer. According to this system, embryos were scored 1 in the case of an elective transfer of one or two embryos (highest score); elective transfer of 3 embryos yielded a score of 2; if two or three embryos had been transferred non-electively, the score was 3, and if only a single embryo was transferred nonelectively, it was scored 4 (worst score).

In our research, we adopted yet another approach. If multiple embryos of different quality were transferred, we assumed that (1) implanting embryo was the best quality embryo (the so-called leading embryo) and (2) the leading embryo determined embryo quality score of the entire group of transferred embryos. The first assumption was well documented in the literature (Hourvitz et al., 2006) and the second one was additionally tested in our sample.

#### **2.2 Relevant studies encompassing the measures of embryo quality**

Despite the fact that important hypothesis blames defective early embryo development for poorer IVF-ET outcome in patients with endometriosis, relatively few studies have analyzed the association between quality of transferred embryo(s) and endometriosis. We will briefly present several important studies on the subject.

A group of Spanish investigators conducted three separate retrospective analyses of the success of their IVF-ET and oocyte donation program in patients and donors with and

Embryo Quality and Pregnancy Outcome in Infertile Patients with Endometriosis 387

A group of Norwegian investigators also confirmed detrimental effect of endometriosis on embryo quality (Tanbo, 1995). They analyzed 215 women (385 cycles) whose main indication for IVF-ET was unexplained infertility (ovulatory women, with patent tubes and normal uterine cavity, normal laparoscopy and normal sperm characteristics), 143 women (285 cycles) with endometriosis as the only indication and 180 women (353 cycles) with tubal infertility (control group). Cleavage rate and failure of cleavage were used as criteria of embryo quality. Significantly lower cleavage rates were observed in both unexplained infertility and endometriosis groups compared with the tubal infertility group. Total failure of cleavage was 19.2% in unexplained infertility, 14.3% in endometriosis and 3.6% in tubal infertility group (p<0.0001). Since there were no differences in sperm characteristics between the groups, the authors speculated that lower cleavage rates could be a consequence of inferior oocyte quality in unexplained infertility

In an interesting study of authors from U.S.A. a total of 235 preimplantation embryos were retrospectively analyzed (Brizek et al., 1995). These embryos were obtained from 56 IVF-ET cycles performed in 30 women. Sixteen patients had endometriosis as the main indication for the procedure and the remaining 14 were controls without endometriosis who were chosen randomly from other diagnosis categories. The incidence of specific phenotypes ranging from normal 2PN zygote to different types of abnormal embryos was then recorded on days 1 and 2 following fertilization. An increased incidence of aberrant development of embryos derived from women with endometriosis was demonstrated. There were three abnormal phenotypes on day 1 and two abnormal phenotypes on day 2 which were significantly more prevalent in patients with endometriosis. However, there was no statistical difference in the number of normal embryos in patients with endometriosis compared to controls on day 1 or day 2. Despite the fact that the effect of endometriosis was observed in the developmental dynamics of the fertilized ovum, no gross endometriosisspecific morphological changes in oocytes recovered from endometriosis group could be

In contrast to previously cited observations, several other studies failed to show negative influence of endometriosis on the parameters of embryo quality. A group of authors from the U.S.A. conducted a retrospective analysis of 284 IVF-ET cycles from patients with a sole diagnosis of endometriosis, or tubal factor, or unexplained infertility (Arici et al., 1996). All of the patients had laparoscopy prior to the IVF procedure. The criteria for the diagnosis of unexplained infertility were confirmed ovulatory cycles, normal tubal patency on hysterosalpingography, normal sperm analyses. The severity of endometriosis was graded as defined by the Revised American Fertility Society classification (The American Fertility Society, 1985) and patients were further divided into two subgroups as minimal to mild (stages I and II) and moderate to severe (stages III and IV). Quality of embryos was assessed on the day of the transfer in line with the system used by the authors' center according to their morphology as observed under the inverted microscope (morphological grades I to V). In the final analysis, the researchers used "the average embryo quality score" for the given subgroup of patients, which was probably calculated as the arithmetical average of all embryo quality scores expressed as grades I to V. No statistically significant difference in "average embryo score" among subgroups were noted (1.8 ± 0.5 in the minimal to mild

and endometriosis group.

seen.

without endometriosis (Pellicer at al., 1995). In the segment of the study on early embryonic development, which was performed on 36 women with endometriosis and on 34 with tubal infertility used as a control group, they explored the embryos grown in vitro for 72 hours before embryo transfer. Embryo quality assessment system included the number of blastomeres and the degree of fragmentation after 48 and 72 hours in culture. If embryos presented only one or two blastomeres 72 hours after oocyte retrieval, it was considered that an embryo arrest had occurred. After 72 hours in culture, there was a significant decrease in the number of blastomeres in endometriosis compared to tubal infertility patients (5.4 ± 0.1 versus 6.1 ± 0.3 blastomeres, respectively, p<0.04) and a significant increase in the percentage of arrested embryos (57.4 ± 2.3 in patients with endometriosis versus 45.2 ± 5.8 in control group, p<0.05). In order to control for the influence of semen parameter on embryo quality, researchers further subdivided groups of patients taking into account the quality of semen. If abnormal semen was used for in vitro fertilization, higher degree of embryonic arrest was observed in comparison to the group with normal semen parameters (55.6 ± 6.4 vs. 20.3 ± 7.9 (p<0.01), respectively, in the group with tubal infertility; 61.8 ± 2.6 vs. 47.5 ± 2.8 (p<0.003), respectively, in the group with endometriosis). Also, if the semen used had normal characteristics, significantly more arrested embryos were noted in patients with endometriosis compared to patients with tubal infertility (p<0.001). Further insight into the problem was attained when the same researchers analyzed pregnancy outcome of oocyte donation with regards to the origin of donated oocytes. This segment of the research incorporated a total of 178 embryo transfers in 141 women. If oocytes were donated by donors without endometriosis, implantation and ongoing pregnancy rates were comparable in both recipients with and those without endometriosis. If oocytes were collected from donors with endometriosis, significantly lower implantation rates were reported in recipients (p<0.05). The authors of this study concluded that infertility in patients with endometriosis may be related to oocyte alterations which result in embryos of lower quality and reduced implantation ability, although the impact of hostile (anti-implantatory) environment on embryos of normal developmental potential cannot be ruled out.

Another study which reported negative impact of endometriosis on embryo quality was the prospective case control study in which researchers included 37 patients with "true" endometriomas and 56 patients without any complex ovarian cysts as controls (Yanushpolsky et al., 1998). All endometriomas were larger than 1 cm in diameter and would be classified as stage III endometriosis according to The ASRM-revised classification of endometriosis (The American Fertility Society, 1985). Only patients with complex "chocolate" cysts in which CA 125 levels in cyst fluid were >100.000 U/ml ("true" endometriomas) were included in the study. Embryo quality was expressed as the number of embryos reaching at least four-cell stage on the second day after oocyte retrieval. Quality of the embryos in the group of patients with endometriomas was significantly reduced compared to controls (p=0.09). Also, in patients with endometriomas, significantly fewer oocytes were retrieved (p=0.06) and early pregnancy loss rate (biochemical pregnancies and early clinical spontaneous miscarriages combined) was significantly higher (p=0.04). Interestingly, fertilization rate and implantation rate were not significantly different between the studied groups.

without endometriosis (Pellicer at al., 1995). In the segment of the study on early embryonic development, which was performed on 36 women with endometriosis and on 34 with tubal infertility used as a control group, they explored the embryos grown in vitro for 72 hours before embryo transfer. Embryo quality assessment system included the number of blastomeres and the degree of fragmentation after 48 and 72 hours in culture. If embryos presented only one or two blastomeres 72 hours after oocyte retrieval, it was considered that an embryo arrest had occurred. After 72 hours in culture, there was a significant decrease in the number of blastomeres in endometriosis compared to tubal infertility patients (5.4 ± 0.1 versus 6.1 ± 0.3 blastomeres, respectively, p<0.04) and a significant increase in the percentage of arrested embryos (57.4 ± 2.3 in patients with endometriosis versus 45.2 ± 5.8 in control group, p<0.05). In order to control for the influence of semen parameter on embryo quality, researchers further subdivided groups of patients taking into account the quality of semen. If abnormal semen was used for in vitro fertilization, higher degree of embryonic arrest was observed in comparison to the group with normal semen parameters (55.6 ± 6.4 vs. 20.3 ± 7.9 (p<0.01), respectively, in the group with tubal infertility; 61.8 ± 2.6 vs. 47.5 ± 2.8 (p<0.003), respectively, in the group with endometriosis). Also, if the semen used had normal characteristics, significantly more arrested embryos were noted in patients with endometriosis compared to patients with tubal infertility (p<0.001). Further insight into the problem was attained when the same researchers analyzed pregnancy outcome of oocyte donation with regards to the origin of donated oocytes. This segment of the research incorporated a total of 178 embryo transfers in 141 women. If oocytes were donated by donors without endometriosis, implantation and ongoing pregnancy rates were comparable in both recipients with and those without endometriosis. If oocytes were collected from donors with endometriosis, significantly lower implantation rates were reported in recipients (p<0.05). The authors of this study concluded that infertility in patients with endometriosis may be related to oocyte alterations which result in embryos of lower quality and reduced implantation ability, although the impact of hostile (anti-implantatory) environment on embryos of

Another study which reported negative impact of endometriosis on embryo quality was the prospective case control study in which researchers included 37 patients with "true" endometriomas and 56 patients without any complex ovarian cysts as controls (Yanushpolsky et al., 1998). All endometriomas were larger than 1 cm in diameter and would be classified as stage III endometriosis according to The ASRM-revised classification of endometriosis (The American Fertility Society, 1985). Only patients with complex "chocolate" cysts in which CA 125 levels in cyst fluid were >100.000 U/ml ("true" endometriomas) were included in the study. Embryo quality was expressed as the number of embryos reaching at least four-cell stage on the second day after oocyte retrieval. Quality of the embryos in the group of patients with endometriomas was significantly reduced compared to controls (p=0.09). Also, in patients with endometriomas, significantly fewer oocytes were retrieved (p=0.06) and early pregnancy loss rate (biochemical pregnancies and early clinical spontaneous miscarriages combined) was significantly higher (p=0.04). Interestingly, fertilization rate and implantation rate were not significantly different

normal developmental potential cannot be ruled out.

between the studied groups.

A group of Norwegian investigators also confirmed detrimental effect of endometriosis on embryo quality (Tanbo, 1995). They analyzed 215 women (385 cycles) whose main indication for IVF-ET was unexplained infertility (ovulatory women, with patent tubes and normal uterine cavity, normal laparoscopy and normal sperm characteristics), 143 women (285 cycles) with endometriosis as the only indication and 180 women (353 cycles) with tubal infertility (control group). Cleavage rate and failure of cleavage were used as criteria of embryo quality. Significantly lower cleavage rates were observed in both unexplained infertility and endometriosis groups compared with the tubal infertility group. Total failure of cleavage was 19.2% in unexplained infertility, 14.3% in endometriosis and 3.6% in tubal infertility group (p<0.0001). Since there were no differences in sperm characteristics between the groups, the authors speculated that lower cleavage rates could be a consequence of inferior oocyte quality in unexplained infertility and endometriosis group.

In an interesting study of authors from U.S.A. a total of 235 preimplantation embryos were retrospectively analyzed (Brizek et al., 1995). These embryos were obtained from 56 IVF-ET cycles performed in 30 women. Sixteen patients had endometriosis as the main indication for the procedure and the remaining 14 were controls without endometriosis who were chosen randomly from other diagnosis categories. The incidence of specific phenotypes ranging from normal 2PN zygote to different types of abnormal embryos was then recorded on days 1 and 2 following fertilization. An increased incidence of aberrant development of embryos derived from women with endometriosis was demonstrated. There were three abnormal phenotypes on day 1 and two abnormal phenotypes on day 2 which were significantly more prevalent in patients with endometriosis. However, there was no statistical difference in the number of normal embryos in patients with endometriosis compared to controls on day 1 or day 2. Despite the fact that the effect of endometriosis was observed in the developmental dynamics of the fertilized ovum, no gross endometriosisspecific morphological changes in oocytes recovered from endometriosis group could be seen.

In contrast to previously cited observations, several other studies failed to show negative influence of endometriosis on the parameters of embryo quality. A group of authors from the U.S.A. conducted a retrospective analysis of 284 IVF-ET cycles from patients with a sole diagnosis of endometriosis, or tubal factor, or unexplained infertility (Arici et al., 1996). All of the patients had laparoscopy prior to the IVF procedure. The criteria for the diagnosis of unexplained infertility were confirmed ovulatory cycles, normal tubal patency on hysterosalpingography, normal sperm analyses. The severity of endometriosis was graded as defined by the Revised American Fertility Society classification (The American Fertility Society, 1985) and patients were further divided into two subgroups as minimal to mild (stages I and II) and moderate to severe (stages III and IV). Quality of embryos was assessed on the day of the transfer in line with the system used by the authors' center according to their morphology as observed under the inverted microscope (morphological grades I to V). In the final analysis, the researchers used "the average embryo quality score" for the given subgroup of patients, which was probably calculated as the arithmetical average of all embryo quality scores expressed as grades I to V. No statistically significant difference in "average embryo score" among subgroups were noted (1.8 ± 0.5 in the minimal to mild

Embryo Quality and Pregnancy Outcome in Infertile Patients with Endometriosis 389

were also analyzed. The secondary goal was to examine the influence of embryo quality on various outcomes of IVF-ET cycles against all other important variables as controls in the

Data used in this analysis were received from the centre's database on couples treated for infertility from 2003 to 2010. If there any data for any variable was missing from the database, the patient's documentation (paper records) was checked. If it was still impossible to find the missing data, the patient was excluded from further analysis. Patients included were under 43 years of age and prior to entering an IVF/ICSI treatment, underwent all tests

The observed cycles were divided into two groups: 173 cycles were performed in patients with endometriosis as the only indication for treatment and 173 cycles in women with tubal factor infertility (control group). The patients from tubal factor group were individually matched with women with endometriosis by age group (<30, 30-34, 35-39, >39 years), number of retrieved oocytes (<5, 5 or more) and number of transferred embryos (1, 2 or 3). Patients were most frequently stimulated according to the protocol involving gonadotrophin-releasing hormone agonists (GnRH-a) (almost exclusively using the long protocol). In the few remaining patients, the protocol with gonadotrophin-releasing hormone antagonists (GnRH-ant) was applied. GnRH agonists used were triptorelin (Diphereline®, Ipsen Pharma Biotech, France), gosereline (Zoladex®, Zeneca Pharmaceuticals, England) or busereline (Suprefact®, Sanofi Aventis, France). Cetrorelix (3 mg) (Cetrotide®, Merck Serono, Switzerland) was used as a GnRH antagonist. Follicle growth was predominantly stimulated by recombinant FSH (Gonal F®, Merck Serono, Switzerland), while human menopausal gonadotrophin (HMG) (Menopur ®, Ferring Pharmaceuticals, Switzerland) was used occasionally. On the day when at least two follicles reached an average diameter of 18 mm, final maturation of the oocyte was stimulated by the urinary human HCG (Profasi®, Merck Serono, Switzerland, using a dose of 10,000 IU) or human recombinant HCG (Ovitrelle®, Merck Serono, Switzerland, 250 mg dose). A detailed description of the laboratory procedures can be found elsewhere (Kovacic et al., 2009). Approximately 36 hours (36 ± 1) following the administration of HCG, oocytes were recovered by ultrasound-guided trans-vaginal follicle aspiration. Fertilization was performed through IVF or ICSI. Medicult® media (MediCult, Denmark) were used for oocyte culturing. Pursuant to the protocol of our centre, only one or maximally two blastocysts were transferred on the fifth, exceptionally on the fourth day following follicle aspiration. Labotec® catheter (Labotec, Germany) was used for blastocyst transfer. In line with the legislation in force at the time of the study, the couple was allowed to decide on the number of embryos to be transferred. Embryos were transferred only after both partners signed the official consent form for the transfer of embryos. A day after the follicle aspiration, all patients started receiving didrogesterone (30 mg/day) (Dabroston®, Belupo, Croatia) or micronized progesterone (600 mg/day) (Utrogestan®, Laboratories Besins

Quality of transferred blastocysts was evaluated by a blastocyst classification system based on morphological criteria, developed by our centre (Kovacic et al., 2004). This classification

prescribed by the protocol for clinical examination of infertile couples.

group of women with endometriosis.

International, France) for luteal support.

**2.3.1 Materils and methods** 

endometriosis group vs. 2.0 ± 0.6 in the moderate to severe endometriosis group vs. 1.9 ± 0.5 in the tubal infertility group vs. 1.8 ± 0.6 in the unexplained infertility group; p>0.05). Surprisingly, when the data were analyzed according to the stage of endometriosis, in the group with moderate to severe endometriosis (stages III and IV) a significantly higher fertilization rate was observed compared to the group with minimal to mild endometriosis (stages I and II) (78.4% vs. 66.8%, respectively; p=0.001). However, implantation rates were low and not significantly different between these subgroups (5.5% in the group with moderate to severe endometriosis vs. 2.8% in the group with minimal to mild endometriosis, p=0.46).

Another study which failed to show negative impact of endometriosis on embryo quality was conducted by Swedish researchers (Bergendal et al., 1998). The analysis included a total of 65 IVF-ET cycles in 48 patients with endometriosis as the only apparent cause of infertility and 98 cycles in 98 patients in whom tubal factor was the only apparent cause of infertility (controls). The embryos were graded according to criteria set by the authors' center (morphology and cleavage stage) with embryo quality scores raging from 1 to 3, with 3 being the best score. The average score of the whole subgroup (defined as arithmetical average of all scores) was used in the final analysis. Despite the fact that fertilization rate was significantly higher in patients with tubal infertility compared to patients with endometriosis (78.3 ± 18.3% vs. 60.1 ± 31.7%, respectively; p=0.00001), no difference was noted in cleavage rates (87.9 ± 19.1% in the tubal factor group vs. 85.2 ± 22.1% in the endometriosis group; p=0.43) or morphological score of embryo for ET (2.5 ±0.39 in the tubal infertility group vs. 2.4 ± 0.4 in the endometriosis group; p=0.45).

In yet another study which reported results on the impact of endometriosis on embryo quality (Dmowski et al., 1995), a retrospective analysis of 237 consecutive IVF-ET cycles in patients with and without endometriosis was conducted. In the group without endometriosis, indications for IVF-ET were tubal disease, pelvic adhesions, male factor, unexplained infertility, ovarian dysfunction ant other factors. In this study, the number of oocytes cleaved was taken as the indirect measure of embryo quality. The authors reported there was no difference between groups in the number of fertilized and cleaved oocytes, but no exact numerical values for these variables were included in the published report. The lack of properly defined control group (endometriosis vs. all other indications) and the absence of further details on development of transferred embryos warrant caution for interpretation, at least in the segment of the study pertaining to embryo quality.

#### **2.3 Our study**

We conducted a retrospective clinical study which encompassed 346 stimulated IVF or ICSI cycles with the transfer of one or two blastocysts performed at the Department of Reproductive Medicine and Gynecological Endocrinology at the University Medical Centre of Maribor, Slovenia.

The primary objective of our study was to examine possible differences in direct and indirect indicators of embryo quality between women with endometriosis as the only indication for the treatment and an adequate control group of women with tubal factor only. Possible differences in various other outcomes of IVF-ET cycles between these two groups were also analyzed. The secondary goal was to examine the influence of embryo quality on various outcomes of IVF-ET cycles against all other important variables as controls in the group of women with endometriosis.
