{"paper_id":"cd65d095-07bb-414a-97fb-ad97e5d1989d","body_text":"Adenomyosis is a benign disease of the uterus characterized by the presence of\nendometrial glands and stroma within the myometrium. 1  Although the results of studies evaluating the effects of adenomyosis on the\noutcome of  in vitro  fertilization–embryo transplantation (IVF-ET)\nhave been controversial, 2  most have reported a negative impact. 3 – 5  Some women with adenomyosis\nthus receive pretreatment with a gonadotropin-releasing hormone (GnRH) agonist\nbefore frozen embryo transfer (FET). 6  However, the effect and duration of pretreatment with a GnRH agonist before\nFET remain controversial. 7 , 8 \nMoreover, long-term pretreatment with a GnRH agonist before FET increases the\nduration and costs of therapy. There is thus a need to identify a noninvasive\nbiological marker to predict the clinical outcome of FET in women with adenomyosis,\nto assess the necessity of pretreatment and predict the optimal timing of hormone\nreplacement therapy (HRT).\nCancer antigen 125 (CA125) is the most common serum marker used in screening for the\npresence and extent of adenomyosis. 9  Sheth and Ray reported that greater enlargement of the uterus due to severe\nadenomyosis was associated with a greater increase in CA125 levels, 10  and Kil et al. 9  reported that the mean serum CA125 level was significantly higher in women\nwith adenomyosis than in patients with myoma. However, the association between serum\nCA125 levels before HRT and the clinical outcome of FET in patients with adenomyosis\nhas not been reported.\nThis retrospective study thus aimed to evaluate the predictive value of serum CA125\nlevels before HRT on the pregnancy outcomes of women with adenomyosis during FET\ncycles.\n\nThis retrospective study included 509 women with adenomyosis undergoing\nIVF/intracytoplasmic sperm injection (ICSI) at the Institute of Reproductive\nMedicine, The First Affiliated Hospital of Sun Yat-Sen University (Guangzhou,\nGuangdong, China), between January 2013 and April 2019. The inclusion criteria\nfor the study were: (i) diagnosis of adenomyosis by transvaginal color Doppler\nultrasonography or magnetic resonance imaging before FET and (ii) age ≤39 years\nat the time of commencement of IVF/ICSI. The exclusion criteria were: (i) the\npresence of hydrosalpinges, intrauterine adhesion, tumor-related disease, pelvic\ninflammatory diseases, or endometriosis, (ii) endometrial thickness <7 mm on\nthe day of transformation before FET, (iii) prior preimplantation genetic\ntesting, and (iv) patient’s partner underwent testicular sperm extraction\nbecause of non-obstructive azoospermia.\nEighty-four patients who underwent a total of 114 cycles of FET were included in\nthe final analysis. Given that serum CA125 levels <35 U/mL were previously reported 11  in more than 95% of healthy women, we divided the included FET cycles\ninto two groups based on a serum CA125 cut-off value of 35 U/L before\nendometrial preparation using HRT: group A had normal CA125 levels (≤35 U/mL,\nn = 70 cycles) and group B had abnormal CA125 levels (>35 U/mL, n = 44\ncycles).\nThis study was approved by the Institutional Review Board of the First Affiliated\nHospital of Sun Yat-Sen University on 11 January 2020 (reference number:\n2020080). The participants were de-identified and the Institutional Review Board\ntherefore waived the need for informed consent.\nAfter ovarian stimulation and oocyte retrieval, embryos were obtained by IVF or\nICSI. The embryos were graded on day 3 or 5 after oocyte retrieval using a\nstandardized scoring system. Embryos that met the eligibility criteria were\nregarded as viable and were subsequently cryopreserved using the vitrification\nfreezing method. The vitrification and thawing procedures were carried out as\ndescribed by Kuwayama et al. 12  Briefly, embryo vitrification was performed using a Cryotop®\nVitrification system (Kitazato Corporation, Tokyo, Japan) with dimethyl\nsulfoxide, ethylene glycol, and sucrose as cryoprotectants. The embryos were\nthawed in decreasing levels of sucrose solution (1, 0.5, and 0 M).\nOver 80% of the included patients received depot GnRH agonist pretreatment before\nHRT for 1 to 4 months, with 3.75 or 1.875 mg triptorelin (Decapeptyl®; Ferring\nPharmaceuticals, Kiel, Germany) per month. The first injection was administered\nduring the early follicular phase of the menstrual cycle. Serum CA125 levels\nwere measured the day before starting the HRT protocol. Oral estradiol valerate\nwas administered at 4 mg/day for 14 days. Patients were monitored by\ntransvaginal ultrasound and blood hormone levels. Endometrial thickness, uterus\nvolume, and type of adenomyosis were recorded during ultrasound monitoring.\nUterine volume (V) was calculated by assuming that it was an ellipsoid, using\nthe formula V = D1×D2×D3 × 0.52, where D1 = transverse diameter, D2 =\nanteroposterior diameter, and D3 = longitudinal diameter. Progesterone was\nadministered if the thickness of the endometrium was ≥7 mm. Day 3 (D3) embryos\nwere transferred on D4 of progesterone administration, and D5 or D6 blastocysts\nwere transferred on D6 of progesterone administration. If the endometrium had\nnot reached a thickness of 7 mm by D15, the dose of estradiol valerate was\nincreased and continued for an additional 3 to 5 days. If the endometrial\nthickness had not reached 7 mm by D20, the cycle was usually cancelled.\nThe same doses of estrogen and progesterone were administered until a serum beta\nhuman chorionic gonadotropin assay was conducted at D14 after FET. If the assay\nresult was positive, HRT was continued until week 10 of the pregnancy.\nImplantation rate was defined as the number of gestational sacs observed on\nultrasonography divided by the number of transferred embryos. Clinical pregnancy\nwas defined as the presence of an active fetal heart detected by ultrasonography\nat 5 to 6 weeks after FET. The miscarriage rate was defined as the number of\nclinical pregnancies lost before 28 weeks of pregnancy divided by the total\nnumber of clinical pregnancies. Ongoing pregnancy was defined as a viable\nintrauterine pregnancy of at least 12 weeks, confirmed by ultrasonography.\nThe Kolmogorov–Smirnov test was used to determine if the continuous variables\nwere normally distributed. Normally distributed data were compared using\nunpaired Student’s  t -tests, and skewed data using the\nMann–Whitney U test. Categorical variables were analyzed using χ 2  or\nFisher’s exact test, where appropriate. Binary logistic regression analysis was\nperformed to detect the association between serum CA125 levels before HRT and\nthe clinical outcomes of FET, while controlling for important confounders.\nReceiver operating characteristic (ROC) curve analysis was used to evaluate the\nability of serum CA125 levels before HRT to predict the clinical outcomes of\nFET. Statistical analysis was performed using IBM SPSS Statistics for Windows\nVersion 23.0 (IBM Corporation, Armonk, NY, USA). A P value ≤0.05 was considered\nstatistically significant.\n\nThe patient selection process is shown in  Figure 1 . Eighty-four patients who underwent\n114 FET cycles were included in the analysis. Adenomyosis was diagnosed by\ntransvaginal color Doppler ultrasonography or magnetic resonance imaging. Serum\nCA125 levels >35 U/mL occurred before 44 cycles of HRT and levels ≤35 U/mL before\n70 cycles.\nFlowchart of patient selection procedures.\nCA125, cancer antigen 125; IVF,  in vitro  fertilization;\nICSI, intracytoplasmic sperm injection; FET, frozen embryo transfer.\nThe baseline demographic and clinical variables of the two groups of patients are\npresented in  Table 1 .\nThere was no significant difference in age at freezing, age at thawing, body mass\nindex, duration of infertility, cause of infertility, fertilization method, previous\nnumber of thawing cycles, developmental stage of the transferred embryos, number of\ntransferred embryos, distribution of GnRH agonist pretreatment, distribution of\ncoexisting endometriosis, type of adenomyosis, or baseline uterine volume between\nthe groups. There was also no significant difference in endometrium thickness,\nestradiol (E2) levels, or progesterone levels between the two groups on the day of\nprogesterone administration. However, the mean serum CA125 level before GnRH agonist\nadministration was significantly higher in the CA125 >35 U/mL group compared with\nthe CA125 ≤35 U/mL group.\nBaseline demographic and clinical variables of patients in relation to cancer\nantigen 125 levels.\nValues given as mean ± standard deviation or n(%).\nCA125, cancer antigen 125; FET, frozen embryo transfer; BMI, body mass\nindex; IVF,  in vitro  fertilization; ICSI,\nintracytoplasmic sperm injection; GnRHa, gonadotropin-releasing hormone\nagonist; E2, estradiol; P, progesterone.\nThe clinical outcomes of the two groups are shown in  Table 2 . There were no significant\ndifferences in implantation rates between the CA125 ≤35 U/mL and CA125 >35 U/mL\ngroups (28.45% vs. 22.89%, respectively). The clinical pregnancy rate was slightly\nhigher in the CA125 ≤35 U/mL group before HRT, but the difference was not\nsignificant (35.71% vs. 31.82%, respectively). Moreover, the ongoing pregnancy rate\nwas also slightly higher in the CA125 ≤35 U/mL group (31.43% vs. 27.27%,\nrespectively) and the miscarriage rate was slightly lower (20.00% vs. 35.71%,\nrespectively), but neither of these results was significant.\nClinical outcome of patients in relation to cancer antigen 125 levels.\nCA125, cancer antigen 125; FET, frozen embryo transfer.\nGiven that pretreatment with a GnRH agonist is an important factor affecting the\npregnancy outcomes of FET, we analyzed the data separately for patients pretreated\nwith a GnRH agonist ( Table\n3 ). Notably, there were no significant differences in the clinical\noutcomes of patients pretreated with a GnRH in relation to CA125 level.\nSubgroup analysis of patients with gonadotropin-releasing hormone agonist\npretreatment before hormone replacement therapy.\nGnRHa, gonadotropin-releasing hormone agonist; CA125, cancer antigen 125;\nHRT, hormone replacement therapy.\nThe two groups still had similar chances of clinical pregnancy after adjusting for\nage, baseline serum CA125 levels, and serum CA125 before FET (adjusted odds ratio\n[OR] = 1.31; 95% confidence interval [CI] = 0.56–3.06), ongoing pregnancy (adjusted\nOR = 1.36; 95% CI = 0.57–3.27), and miscarriage (adjusted OR = 0.48; 95%\nCI = 0.08–2.76). Patients pretreated with GnRH agonist also had similar chances of\nclinical pregnancy (adjusted OR = 1.16; 95% CI = 0.45–2.95), ongoing pregnancy\n(adjusted OR = 1.20; 95% CI = 0.45–3.22), and miscarriage (adjusted OR = 0.92; 95%\nCI = 0.12–7.35), irrespective of CA125 level. The results of the binary logistic\nregression analyses are shown in  Tables 4  and  5 .\nLogistic regression analysis of pregnancy outcomes in relation to cancer\nantigen 125 levels.\nAnalysis adjusted for age, baseline serum CA-125 and serum CA-125 before\nfrozen embryo transfer.\nCA125, cancer antigen 125; FET, frozen embryo transfer; OR, odds ratio;\nCI, confidence interval.\nLogistic regression analysis of pregnancy outcomes in patients with\ngonadotropin-releasing hormone agonist pretreatment in relation to cancer\nantigen 125 levels.\nAnalysis adjusted for age, baseline serum CA-125 and serum CA125 before\nfrozen embryo transfer.\nGnRHa, gonadotropin-releasing hormone agonist; CA125, cancer antigen 125;\nFET, frozen embryo transfer.\nThe area under the ROC curve (0.474) indicated that CA125 levels had no predictive\nvalue for the outcome of clinical pregnancy ( Figure 2 ).\nReceiver operating characteristic (ROC) curve for serum levels of cancer\nantigen 125 before hormone replacement therapy as predictor of clinical\npregnancy among patients with adenomyosis undergoing frozen embryo transfer\ncycles.\n\nTo the best of our knowledge, this study provides the first evidence qualifying the\nimpact of adenomyosis on pregnancy outcomes of FET, based on serum CA125 as a\nbiological marker. The results demonstrated that CA125 levels before HRT had no\nprognostic significance on the outcome of FET.\nThe extent of adenomyosis has previously been associated with reproductive outcomes, 13  and more severe adenomyosis was associated with a greater increase in serum\nCA125 levels. 10  However, adenomyosis may be associated with numerous conditions that could\nimpair embryo implantation. The junctional zone of myometrial activity was reported\nto be affected by adenomyosis 14 , and research also revealed that abnormal contractile activity of the\njunctional zone in patients with adenomyosis was associated with lower implantation\nand pregnancy rates following IVF-ET. 15  Moreover, vascularization of the endometrial stroma was unexpectedly\nincreased in patients with adenomyosis, with negative effects on embryo implantation. 16  Furthermore, changes in expression profiles of cytokines and growth factors\nin the endometrium have been related to adenomyosis-associated infertility. 17  These studies suggest that a mere increase in serum CA125 levels is not an\nappropriate measure reflecting the complex influence of adenomyosis on the clinical\noutcome of FET. Furthermore, serum CA125 is a less reliable marker in premenopausal\nwomen because of increases in response to various conditions, such as endometriosis,\nadenomyosis, tumor formation, and even menstruation, 18  and an irrelevant increase in serum CA125 levels could result in\nmisdiagnosis.\nThe results of subgroup analysis showed that serum CA125 levels before HRT were not\nassociated with the clinical outcome of FET in patients pretreated with a GnRH\nagonist. Xie et al. 19  reported that serum CA125 levels were significantly reduced after long-term\ntreatment with a GnRH agonist in patients with adenomyosis, and Niu et al. 20  found that long-term pituitary downregulation before FET improved pregnancy\noutcomes in these women. Lower serum CA125 levels could be associated with shrinking\nof the uterus and milder pelvic adhesions. 10 , 21  However, the reasons for the improved pregnancy outcomes in these patients\nare complicated and not well understood. 22 , 23  Thus, a decrease in serum CA125 levels before HRT is not predictive of the\nclinical outcome of FET in patients with adenomyosis.\nThere were some limitations to this study, including the retrospective nature of the\nstudy and the relatively small sample size, which could cause bias. Further\nprospective studies with larger cohorts are therefore required to verify the results\nof this study.\nIn conclusion, the results of our study suggest that serum CA125 levels before HRT\nare not related to the rates of implantation, clinical pregnancy, ongoing pregnancy,\nor miscarriage following FET in women with adenomyosis. Sole detection of serum\nCA125 levels before HRT is thus not a valid measure, resulting in unnecessary cost\nand increased anxiety for the patient. The combined detection of other biological\nmarkers and/or the identification of novel markers is required to increase the\npredictive accuracy in the future.","source_license":"CC0","license_restricted":false}