{"paper_id":"4136f3a7-b2ed-498a-9e91-8de073710d60","body_text":"Endometriosis is an enigmatic disorder affecting 10-15% of females of reproductive\nage. It is estimated that up to 40% of women undergoing laparoscopic investigation\nfor infertility have endometriosis ( Yilmaz\n et al ., 2021 ). Endometrioma may be present in 17-44%\nof women diagnosed with endometriosis ( Alshehre\n et al ., 2021 ;  Hoyle\n& Puckett, 2022 ). Endometriosis is a debilitating disease and has a\nconsiderable economic burden on patients and the society ( Esmaeilzadeh  et al ., 2015 ;  Parasar  et al ., 2017 ).\nClassification of endometriosis has been established by the American Society for\nReproductive Medicine (ASRM), and it can be categorized into four stages: I\n(minimal), II (mild), III (moderate), and stage IV (severe) ( Mirabi  et al ., 2019 ). More advanced stages may\nbe deeply invasive and present as endometrioma that can lead to specific\ncomplications, such as decreased ovarian reserve in these patients ( Hoyle & Puckett, 2022 ).\nStill, endometriosis is frequently reported as the underlying etiology of\nsubfertility, so assisted reproductive technology (ART) is the common choice for\npatients with endometriosis-associated sterility who wish to conceive. Moderate and\nsevere stages of endometriosis usually require  in vitro \nfertilization (IVF) ( Alshehre  et\nal ., 2021 ). Whether endometriosis contributes to infertility has\nlong been debated, and underlying mechanisms resulting from the presence of disease\nand classified by stage possibly affecting fertility potential are poorly known,\nalthough inflammation and reactive oxygen species are believed to contribute\nsignificantly ( Rogers  et al .,\n2017 ).\nResearch findings showed that endometriosis patients may have endocrine and ovulatory\ndisorders, including impaired folliculogenesis compromised granulosa cell and\nfollicle immune homeostasis, premature luteinizing hormone (LH) surges and luteal\nphase defects ( Hamdan  et al .,\n2015 ;  González-Comadran  et\nal ., 2017 ).\nSome investigators strongly emphasize that oocytes from endometriosis patients have\nreduced competence, and high stages of endometriosis consistently lead to reduced\noocyte yield ( Horton  et al .,\n2019 ). Clinical IVF/ICSI studies have investigated antral follicle count,\nnumber and quality of retrieved oocytes and embryos, cycle cancellation and\nfertilization rates in endometriosis patients have either not found significant\ndifferences ( Hamdan  et al .,\n2015 ;  González-Comadran  et\nal ., 2017 ) or detected meaningfully diminished ovarian\nreserve, oocyte yield and number of mature oocytes ( Xu  et al ., 2015 ;  Horton\n et al ., 2019 ;  Orazov\n et al ., 2019 ;  Alshehre  et al ., 2021 ).\nA recent retrospective cohort study stated that the number of antral follicle count\n(AFC) and mature oocytes were significantly lower in infertile women with\nendometrioma, whereas numbers of embryos achieved, clinical pregnancy rates and live\nbirth rates were similar between endometriosis patients and control groups ( Yilmaz  et al ., 2021 ). On the\nother hand, some studies have reported an inverse relationship between different\nstages of endometriosis and ART outcome. They stated that advanced endometriosis has\na deleterious and sustained effect on ovarian reserve and fertility parameters\n( Kuivasaari  et al .,\n2005 ;  Pop-Trajkovic  et al .,\n2014 ;  Orazov  et al .,\n2019 ).\nLi  et al . (2020) showed a negative effect of advanced endometriosis\non cumulative clinical pregnancy per oocyte retrieval cycle, also a systematic\nreview and meta-analysis of the available comparative and observational data\nsuggested that a progressive reduction in ART outcomes has been shown in\nendometriosis-affected patients with increasing disease severity ( Horton  et al ., 2019 ).\nThis finding is contrary to previous systematic reviews and meta-analyses, which\nstated that there was no relevant difference in the chance of achieving clinical\npregnancy and live birth following ART when comparing Stage-III/IV with Stage-I/II\nendometriosis ( Barbosa  et al .,\n2014 ). However, the quality of most of the studies in this systematic\nreview has been very low to moderate. Anyway, reports evaluating the impact of\nendometriosis and different stages of it on the outcomes of IVF/ICSI seemed\ncontroversial, in relation to the focus on different specific outcomes. As yet,\nseveral questions remain unanswered. There is no robust data to recognize the\nplausible negative impact of various stages of endometriosis on the main outcomes of\nART (number of AFC, oocyte competence, embryo development and fertilization rate),\nespecially for limited (Stage-I/II) disease. For these reasons this retrospective\nstudy was conducted to investigate whether the presence and/or the severity of\nendometriosis affects ICSI outcomes, including oocyte quality, fertilization rate\nand embryo quality.\n\nThis investigation was a 1:1 retrospective cohort study of patients undergoing\nfirst-attempt ICSI treatment at the Fatemezahra Infertility and Reproductive Health\nResearch center, Babol, Iran. The study performed in compliance with the\nInstitutional Review Board at Babol University of Medical Sciences\n(NO.IR.MUBABOL.HRI.REC.1398.296) between November 2019 and December 2020 - STROBE\n(Strengthening the Reporting of Observational studies in Epidemiology) guidelines\nfor observational studies were followed to conduct this study.\nAll data were obtained from databank and medical records of infertile patients who\nreferred to our infertility center. This center is one of the best equipped\ndiagnostic and therapeutic infertility centers, and it is a referral center for\ninfertility problems in the north of Iran.\nThe eligibility criteria were <40 years of age, first ICSI, normal sperm in\nthe male and all had previously undergone diagnostic laparoscopy and\ntransvaginal ultrasound using (5 MHz probe Fokuda, Japan). The exclusion\ncriteria were polycystic ovaries, immunological disease, uterine abnormalities,\nprevious history of ovarian surgery, premature ovarian failure and severe male\nfactor according to WHO criteria. Infertile women with laparoscopic and\nultrasound confirmation of endometriosis based on the inclusion criteria were\nselected as the study group, and women with tubal factor diagnosis or\nunexplained infertility were included as the control group. Endometriosis was\nstaged according to the ASRM 1996 classification (based on laparoscopy) ( Mirabi  et al ., 2019 ).\nTo stimulate the follicles, ovulation induction protocol using agonists GnRH\n(Superfact, Aventis Pharma Deutschland, Germany) was started in the mid-luteal\nphase of the cycle (21-23 of the cycle). For initiating, a thin dense\nendometrium < 4 mm, and the absence of any growing follicle > 6 mm should\nbe visualized in the transvaginal ultrasonography (TVS). If the endometrial line\nwas thin in TVS (< 5mm) 14 days after the agonist started, we did controlled\novarian hyperstimulation (COH) using recombinant human follicle-stimulating\nhormone (Cinnal-f, Cinagen-Iran ). Cinnal-f dose was chosen for each patient on\nthe basis of age, day 3 FSH and AMH level and AFC if the endometrial line was\nnot thin in the TVS, superfact continued for two following weeks. When the\nendometrial line reached <5mm, the stimulation was initiated.\nIf not, the cycle was canceled, but the patient did not drop out of the study.\nFollicle’s monitoring was performed 7 or 8 days later and every 2 to 3 days\nthrough TVS to trace the growing follicles. Cinnal-f was added according to\novarian response. Human Chorionic Gonadotropin 10,000 IU (HCG, Daroupakhsh,\nIran) was administrated when at least two oocytes >16 mm were seen. Oocytes\nwere retrieved through TVS-guided 34-36 hr following HCG trigger.\nCollected oocytes were classified into Metaphase II (MII), metaphase I (MI) and\nGerminal vesicles (GV). Embryos were graded morphologically.\nEmbryos with little or no fragmentation and a zona pellucida not extremely thick\nor dark in appearance were classified as Grade A, embryos with equally-sized\nblastomeres, minor cytoplasmic fragmentation covering ≤10% of the embryo\nsurface Grade-B and blastomeres of distinctly unequal size and\nmoderate-to-significant cytoplasmic fragmentation covering >10% of the embryo\nsurface were Grade C ( Mirabi  et\nal ., 2017 ). One or two embryos were transferred per\ntransfer cycle. In each patient, 2 grade-A embryos were transferred on day 2 or\n3.\nBaseline characteristics of participants, ovarian reserve biomarkers (AMH, FSH,\nand AFC), the number and quality of oocytes and embryos and fertilization rate\nwere analyzed and compared between various severity grades of endometriosis and\ncontrol group.\nAll statistical analyses were performed using a software (SPSS 21.0 version).\nKolmogorov-Smirnov test was used to evaluate the normality of variable\ndistribution. The Continuous variables with normal distribution were expressed\nas the mean±SD or as percentages when required. Comparison of demographic\nand clinical characteristics in the various severity grades of endometriosis and\nthe control group were performed with one-way analysis of variance (ANOVA), with\nBonferroni adjustment. The mean difference (MD) and their 95% confidence\ninterval (CI) were calculated for the studied outcomes. Results were also\ncompared between patients with Stage I/II and Stage III/IV endometriosis and the\ncontrol group. Effect sizes were expressed as Eta Squared\n( η 2 ) to show the relative magnitude of\nthe differences between means (Small = 0.01. Moderate = 0.06 or Large = 0.14)\n( Lakens, 2013 ).\n\nTwo hundred and ninety women were diagnosed with endometriosis. Thirty-eight were\nexcluded for different reasons (10 patients had been diagnosed with severe male\nfactor; 2 with premature ovarian failure; 8 with uterine abnormality and 18 patients\nhad polycystic ovaries) ( Fig. 1 ). The data of\n32 patients were not appropriate for analysis, therefore; they were also excluded\nfrom the study.\nFigure 1 Flow of participants through the study.\nFlow of participants through the study.\nOf the 220 endometriosis patients, 48 (21.8%) had ASRM stages I endometriosis, 76\n(34.5%) stage II, 53 (24%) stage III and 43 (19.5%) stage IV. Two hundred and twenty\nwomen with unexplained infertility or laparoscopically diagnosed tubal factor\ninfertility (without any evidence of endometriosis) were included as the control\ngroup.\nThe mean age was 32.4 ± 5.16 years in the endometriosis group and\n31.44±5.64 in the control group ( p =0.25). The mean\n ± SD body mass index of the endometriosis participants was\n25.50±3.0kg/m 2 \n versus  25.95±4.35. Therefore, most of the participants were\noverweight, however, differences between groups were not statistically significant.\nTherefore, if BMI is a factor influencing ART outcomes, this effect is the same\nbetween the groups ( p =0.26). Dyspareunia and infertility duration\nwere higher in the women diagnosed with endometriosis than controls, and this\ndifference was significant. There were no statistically significant differences in\nterms of dysmenorrhea, type of infertility (primary or secondary) and abortion\nbetween groups ( Table 1 ).\nCharacteristics and hormonal assay results of study population.\nThe plasma levels of FSH and prolactin were significantly higher in the endometriosis\ncompared with tubal and unexplained groups ( p <0.001); however,\nthere no significant differences between groups according to the levels of AMH;\nalso, the total duration of gonadotropin stimulation was not statistically\nsignificant between the groups ( Table 2 ).\nHormonal assay results and ICSI outcomes in endometriosis and control\ngroups.\nOn the other hand, the oscillations of FSH and prolactin levels varied in relation to\nthe stage of endometriosis progression. As to ANOVA test, FSH and prolactin levels\nwere significantly higher in stages III/IV compared with controls\n( p <0.001) ( Table\n3 ).\nICSI data and clinical outcome of patients with different stages of\nendometriosis and control groups.\nDespite similar levels of serum AMH between the groups, fewer oocytes in women with\ninfertility associated with endometriosis were retrieved than in patients in the\ncontrol group  (p <0.001). Also, we found a statistically\nsignificant decrease in the number of mature oocytes (MII) in endometriosis\npatients, compared with the control group  (p <0.001).\nANOVA-repeated measures revealed that patients with advanced endometriosis (III/IV)\nhad significantly fewer retrieved oocytes with small effect size\n( p <0.001, η 2 =0.04), lower metaphase II\n oocytes  ( p <0.001,\nη 2 =0.09) and fewer total numbers of embryos\n( p <0.001, η 2 =0.11) compared with less severe\ndisease or women with tubal factor infertility. The effect size was medium. The\nfertilization rate in women with severe endometriosis was similar to that of the\ncontrol group and in those with minimal /mild endometriosis\n( p =0.187).\nPregnancy rates were significantly different in various stages of endometriosis, as\npatients with severe endometriosis had a lower pregnancy rate compared to less\nsevere disease ( p =0.02).\n\nOver the  past two decades , the detrimental influence of\nendometriosis on ART outcomes has been debated in the literature. There is\ncontroversy regarding the impact of different stages of endometriosis on assisted\nreproductive technique outcomes ( Yilmaz  et\nal ., 2021 ).\nAccording to our data, advanced endometriosis had a negative influence on ART\noutcomes. In the current study, the number of retrieved oocytes, number of MII\noocytes and good-quality embryos were decreased in relation to the endometriosis\nprogression. Though we didn’t find considerable differences in the requirement of\ngonadotrophin with increasing severity of endometriosis.\nElevated prolactin levels were also detected in women with endometriosis,\nirrespective of the stage of the disease. This study supports evidence from previous\nobservations by  Mirabi  et al. \n(2019)  and Esmaeilzadeh  et al . (2015), who reported\nprolactin levels act as a probable prognostic biomarker to detect endometriosis\nstages III/IV  vs . I/. The true mechanisms of action of\nhyperprolactinemia in patients with endometriosis are still not fully understood.\nHowever, studies only suggested prolactin concentration progressively increased from\nstage I to IV ( Mirabi  et al .,\n2019 ).\nAlmost all aspects of ART are negatively influenced by severe endometriosis, from the\novarian response during gonadotropin stimulation to pregnancy. The only exception\nwas the fertilization rate. These results are consistent with data obtained in a\nsystematic review and meta-analysis of 8 published studies. They found that severe\nendometriosis can significantly reduce the total number of oocytes, MII oocytes\nretrieved and total high-quality embryos; however, it does not seem to adversely\nimpact fertilization rates ( Alshehre  et\nal ., 2021 ). Also,  Li\n et al.  (2020)  revealed that despite the number of\nmature oocytes and viable embryos are meaningfully lower; there were no\nstatistically significant differences between advanced endometriosis and the\ncomparison group with respect to fertilization rate and pregnancy outcomes.\nKuivasaari  et al . (2005) suggested women with advanced\nendometriosis had worse IVF/ICSI results including fertilization and pregnancy rates\ncompared with women with milder forms of endometriosis or women with tubal\ninfertility.\nIn addition,  Barnhart  et al .\n(2002)  found detrimental effects of advanced endometriosis on developing\nfollicles, oocyte quality and embryo yield, also they reported lower pregnancy and\nlive birth rates compared to minimal and mild endometriosis.\nAlthough in our study the amount of AMH was not meaningfully different between the\ngroups, the higher levels of FSH in some patients and a diminished number of total\nand mature oocytes in advanced endometriosis compared with less severe endometriosis\nenables us to speculate that progression of disease per se is associated with\nreduced ovarian reserve, but this does not translate into better fertilization\nrates, which were the same as in women with the most severe forms of disease\ncompared to women with milder forms of endometriosis or other causes of\ninfertility.\nPrevious studies have suggested that a considerable increase in fertilization failure\nin IVF is much rather due to the sperm-egg interaction in advanced endometriosis. It\ncould also be related to a lower rate of MII oocytes ( Shebl  et al ., 2017 ). Also, the peritoneal\nfluid of endometriosis patients may influence sperm-binding ability and sperm\nmotility. Hence, fertility rates are generally reduced. That’s the reason why ART\nscientists recommend that IVF should not be the first treatment of choice in\nadvanced endometriosis ( Hull  et\nal ., 1998 ;  Shebl  et\nal ., 2017 ;  Li  et\nal ., 2020 ).\nAll patients in the current study underwent ICSI treatment; thus, fertilization rates\nwere the same in advanced endometriosis compared to mild and moderate grades.\nPatients with advanced stages of endometriosis usually receive ovarian surgery,\nalthough post-operative having of ovarian reserve deterioration, postulated by\nclinical evidence ( Chiang  et al .,\n2015 ).\nDespite the plethora of studies on endometriosis, it is not easy to draw any\n definite conclusions  due to the methodological heterogeneity,\nselection bias of studies, differences in the surgical methods, selected outcomes\nand low sample size of studies. Although the women included in our study had no\nprevious history of ovarian cyst excision or resection, we found that advanced stage\nendometriosis  adversely affects  ovarian response and oocyte\nperformance. In fact diminished ovarian reserve is diagnosed as the reduced capacity\nof the ovaries to create oocytes in both quantity and quality ( Zangmo  et al ., 2016 ).  Most studies\n( Aboulghar  et al .,\n2003 ) on severe  endometriosis  usually focus on patients\nwith a previous endometriosis surgery and concluded that patients with a history of\nprevious surgery showed lower responses to gonadotropin stimulation. Also, they had\nless fertilization rates. Hence; there is still uncertainty on the impact of various\nstages of endometriosis on IVF-ICSI outcomes.\nDue to the lack of surgical history in our participants, it seems that advanced\nendometriosis without surgical intervention may be one of the critical factors that\nhave a negative effect on ovarian reserve and ovarian response to stimulation. In\nline with our findings, in a case-control study,  Hock  et al . (2001)  found diminished ovarian reserve in\nstage III/IV endometriosis and concluded this is in accordance with progressive loss\nof ovarian reserve in patients with advanced endometriosis independent of age.  Maneschi  et al.  (1993)  also\nreported a reduced number of developing follicles and vascular activity before any\noperation among infertile women with severe endometriosis, suggesting that the\ndisease may be detriment to the ovary. However, in most studies fertilization rate\nwas not impaired.\nContrary to our results and aforementioned studies, Pop-Trajkovic  et\nal . (2014) reported that fertilization rates in women with severe\ngrades of endometriosis was higher than in those with minimal/mild endometriosis. We\ndo not know  the reason  of  higher fertilization rates among\nstage  (III/IV) endometriosis patients in that  study .\nThis observation may support the hypothesis that the burned-out lesions in women\nwith advanced endometriosis is inactive and causes adhesions in the pelvis, while a\nmilder form of endometriosis is associated with active endometrial glands ( Pop-Trajkovic  et al. ,\n2014 ).\nOn the other hand, several cofactors, such as age, BMI and ovarian reserve may be\nresponsible for the poor IVF/ICSI outcomes in these patients. Based on the\nliterature, the impact of diminished ovarian reserve becomes more pronounced in\npatients of older age whose declining egg quality is associated with higher embryo\naneuploidy ( Ata  et al ., 2019 ;\n Pirtea & Ayoubi, 2020 ). Women in an\naforementioned study were younger than our participants, and most of them had normal\nweight, while our patients were older and half of them were overweight - which may\nbe one reason for the observed higher rates of fertilization.\nMost importantly, patients with advanced stages of endometriosis had meaningfully\nlower viable embryo rates per oocyte retrieved, which suggest the lack of oocyte\ncompetence. Additionally, Patients with severe endometriosis had a lower pregnancy\nrate compared to less severe diseases which is in agreement with other studies\n( Kuivasaari  et al. ,\n2005 ). However, effect size was small and it was meaningless. A systematic\nreview and meta-analysis demonstrated that clinical pregnancy rates are not\nrelevantly different between patients with advanced endometriosis and those with\nmilder forms of endometriosis. Nevertheless, despite the very large number of\nincluded studies and the higher risk of bias in observational studies, they were\nconsidered of very low quality and their results were inconsistent ( Barbosa  et al ., 2014 ).\nOne of the strengths of this study was that all of the participants had\ndiagnostic laparoscopy and the severity of the disease was determined, which\nreduced the risk of misclassification. Since the stage of endometriosis, was\nreported in the data bank of the infertility center, the impact of various\nstages of endometriosis on ART outcomes was completely ascertained from this\nanalysis. The present study has a few limitations such as our study was a\nsingle-center analysis and it had a retrospective design.\n\nBased on the results of this study, we suggest that the high stage of endometriosis\nmay damage ovarian reserve, and it has a detrimental effect on ovarian response\nduring gonadotropin stimulation. We found that patients with severe endometriosis\nhave a trend toward worse outcomes, however; lower ovarian reserves are not\nassociated with lower fertilization and pregnancy rates. Moreover, fertility\nparameters and reproductive outcomes of stages III/IV and stage I/II endometriosis\nseem to be comparable. This finding may help clinicians find appropriate advanced\nendometriosis management.","source_license":"CC0","license_restricted":false}