A significant number of subfertile women with endometriosis may eventually require Artificial Reproductive Techniques (ART) with in-vitro fertilisation (IVF) and/or intra-cytoplasmic sperm injection (ICSI) to conceive. Up to 15% of women undergoing ART have endometriosis (Senapati et al., 2016). Endometriosis is known to be associated with subfertility although its pathophysiology is still unclear. Endometriosis can be detrimental to fertility at the level of the endometrium, oocyte and/ or embryo (Filippi et al., 2014, Reinblatt et al., 2011b).
Research from our group and others showed that women with endometriosis have a lower number of oocytes retrieved per cycle of ART (Hamdan et al., 2015b, Barnhart et al., 2002, Hamdan et al., 2015a, Harb et al., 2013, Lin et al., 2012), and those with endometrioma require a higher dose of FSH stimulation and have a higher cancellation rate during their IVF cycles (Hamdan et al., 2015a). Animal studies suggest that oocyte and embryo development were poorer when exposed to follicular and peritoneal fluid of women with endometriosis (Da Broi et al., 2014, Mansour et al., 2010, Hamdan et al., 2016). Studies on oocyte recipient cycles showed poorer reproductive outcome in recipients who received oocytes from women with endometriosis, thus, strongly suggesting that the oocyte quality of women with endometriosis is compromised (Diaz et al., 2000, Simon et al., 1994), although the vast majority of previous studies directly examining the impact of endometriosis oocyte and embryo quality and development fail to conclude that endometriosis detrimentally impact on oocyte and/or embryo quality as observed in ART cycles (Filippi et al., 2014, Reinblatt et al., 2011a, Dong et al., 2013, Al-Fadhli et al., 2006, Lin et al., 2012, Mekaru et al., 2013, Suzuki et al., 2005). The oocytes from women with endometriosis have structurally and functionally abnormal mitochondria (Xu et al., 2015) and a higher rate of aneuploidy (Gianaroli et al., 2010) compared to controls, although reports on the aberrant morphology of oocyte meiotic spindle are controversial(Mansour et al., 2009, Rajani et al., 2012) .
The current available studies examining the oocyte/embryo quality for women with and without endometriosis suffer from the following limitations. Embryo morphological assessment (Munne et al., 2007) is traditionally subjected to a high intra- and inter-observer variability when considering factors associated with morphological dysmorphisms such as fragmentation, asymmetry, and multinucleation despite the recent consensus on standardising embryo assessment(Alpha Scientists in Reproductive and Embryology, 2011). Only one study utilised a standardised objective embryo morphological assessment (Filippi et al., 2014, Alpha Scientists in Reproductive and Embryology, 2011) Most studies made their assessments on D3 rather than D5 embryo assessment and none of the studies utilised data obtained from time-lapse technology. The majority of the studies assessed the embryo quality as a secondary outcome rather than as their primary outcome.
This study aims to assess the oocyte and embryo development based on recently performed ART cycles, using standardised criteria for the assessment of embryo development, to evaluate the quality of oocytes and embryos in with endometriosis compared to those with no endometriosis.
This study was a retrospective case control study, performed in two tertiary referral fertility centres in the United Kingdom. Anonymised data from IDEAS V.6™ (Electronic IVF database, Mellowood Medical, USA) from January 2011 to December 2014 in both fertility centres were extracted, analysed, and reviewed. Data obtained from the database were exported to Excel (Microsoft, USA) before data analysis was performed using statistical analysis package.
This study included women under 40 years old who underwent IVF treatment using their own gametes. Oocyte donor cycles (altruistic or sharer) or couples with co-existing male factor subfertility that required intra-cytoplasmic sperm injection (ICSI) was excluded.
Study group (Endometriosis, EN) consisted of women who were subfertile for at least 1 year with endometriosis, diagnosed laparoscopically and/or sonographically, irrespective of the disease severity and duration. All the women who had laparoscopically diagnosis and treatment of endometriosis were collectively included in the study group regardless of the type of surgical treatment performed.
Our control groups included women with tubal factor (TF) subfertility or unexplained subfertility (UE) defined as being subfertile for at least 1 year with no identified reasons for subfertility (normal hormonal profile, patent tube(s), and normozoospermia). Women were considered to be in the tubal factor group (TF) if they were subfertile for at least 1 year with evidence of tubal disease in either one or both tubes. If the tubes were blocked resulting in hydrosalphinges, the normal practice in the UK would be to remove or clip the tube(s). They do not have a history of endometriosis, with the absence of any current signs or symptoms of endometriosis, and have no ultrasound evidence of endometriosis.
Ovarian stimulation protocols were limited to the antagonist cycle and long agonist cycle. Short flare agonist cycles were excluded. There was no restriction on the type of stimulation drugs used; however recombinant FSH was the predominant drug of choice, with a starting dose based on the age and AMH level, in accordance to local dosing protocol. The day of trigger (HCG) was determined by the presence of 3 or more follicles over 17 mm. The number of oocytes on the folliculogram > 14 mm was an arbitrary level of cut-off taken to compare the oocyte retrieval rates between the two groups from follicles that were most likely mature at the time of oocyte collection. This is of particular interest because work from our group and others have shown possibility of oocyte DNA damage as a sequelae of endometriosis which impacts on follicular maturation and development (Hamdan et al., 2016, Mansour et al., 2010).
Qualified and trained embryologists from each centre performed the oocytes and embryo assessment. Following oocyte retrieval procedure, oocytes were incubated in the culture media until the in vitro fertilisation procedure. Where possible, the collected oocytes were scored according to the appearance of cumulus cells, zona pellucida, and cytoplasm (Alpha Scientists in Reproductive and Embryology, 2011).
Irrespective of the oocytes score, insemination was performed at approximately 1400H in the afternoon of the same day of egg collection. Fertilisation check was done 17 ± 1 hours post insemination (HPI). Normal fertilisation was defined as presence of 2 polar bodies with two centrally located pronuclei that are evenly sized.
At 68 ± 1 HPI, all embryos were reassessed and scored using standardised scoring system(Alpha Scientists in Reproductive and Embryology, 2011) according to the cell number, fragmentation rate, and multinucleation. The scoring was documented in the embryology record electronically. At this stage, fertilised oocytes that have not cleaved were categorised as arrested at D1 and were discarded.
At D3, selected women underwent embryo transfer procedure, which was decided according to the respective unit’s embryo transfer criteria and protocol. Embryos that were not transferred at D3 were cultured to blastocyst stage. At D5 of culture, embryos were rescored. Suitable good quality blastocysts were usually frozen on the same day or the day after (D6) if the criteria were met.
Time-lapse embryo assessment
As an alternative to conventional daily time-specific embryo morphological check, Eeva™ was used for embryo monitoring following fertilisation check. Eeva™ uses non-invasive, time-lapse imaging of embryos during undisturbed incubation, which detects critical and subtle differences in cell division. The time-lapse data was available only in one of the two centres.
Number of oocytes retrieved is defined as the total number of oocytes collected per oocyte collection procedure. Percentage of oocytes per mature follicles is defined as the total number of oocytes collected over total number of follicles (measuring > 14 mm on the day of trigger). The number of fertilised oocytes is defined as the number of normally fertilised oocytes per number of patient. Fertilisation rate is defined as the number of normally fertilised oocytes per number of inseminated oocytes. D1 arrested embryo is defined as fertilised oocytes in which development has arrested and has not reached cleavage stage. At Day 3, individual embryo was scored into 4 Grades (1, 2, 3, and 4). Grade 1 and Grade 2 embryos at day three were grouped and deemed as poor quality oocytes, whereas Grade 3 and Grade 4 were grouped and deemed as good quality oocytes. Number of embryo transferred and cryopreserved are define as number of embryo transferred into the uterus and frozen respectively, over number of patients. Utilisation rate is defined as the percentage of embryos transferred or cryopreserved, over the number of oocytes retrieved. Blastulation rate is defined as the percentage of blastocysts over the number of fertilised oocytes. Fully expanded and hatching blastocyst (FEHB) is defined as good quality blastocysts at Grade 4 and above. Clinical pregnancy is defined as the presence of at least one fetal heart. For time-lapse data, the time taken for the fertilised embryos to develop from 2-pronuclei stage to 2 cell (P2) and 3 cells (P3) were recorded.
Statistical analysis was performed using unpaired t-test for parametric data. Fischer exact test or chi square test was used for categorical data. A p-value < 0.05 was considered to be significant. One-way ANOVA was used for comparison of more than 2 groups with the normally distributed data. If the ANOVA showed statistically significant difference, post hoc analysis was done using Tukey’s post-hoc test. Kruskal-Wallis test was used for comparison of more than 2 groups with non-parametric data. All statistical analyses were performed using the Statistical Package for Social Sciences software, version 18 (SPSS Inc., Chicago, IL, USA) or Prism6 (GraphPad, USA) except effect size, which was calculated using the Cohen d Test (G*Power, USA). Data is expressed as mean ± standard deviation (SD), or standard error (SE) or as percentage ± SD or SE.