{"paper_id":"abde88b0-87be-4622-aaac-ebcc1874e886","body_text":"Cryopreserved-thawed embryo transfer began\nin 1983 and became a popular, vital component of\nassisted reproduction technology ( 1 ). The transfer\nof a frozen embryo enhances the cumulative pregnancy\nrate, decreases cost, Is easy to perform\nand can be fulfilled successfully in a relatively\nshorter time span in comparison with repeated\nfresh cycles ( 2 - 5 ). Furthermore, endometrial\nreceptivity can be compromised by controlled\novarian hyperstimulation (COH) protocols ( 6 )\nand secretory endometrial transformation ( 7 ).\nEndometrial development in frozen-thawed cycles\ncan be controlled more than during COH\ncycles ( 8 ).\nVarious protocols (gonadotropin/GnRH agonists,\nclomiphene citrate, or exogenous estrogen\nand progesterone) have been discussed in literature\nreviews with regards to the endometrium\npreparation for frozen-thawed embryo transfer ( 3 ,\n 9 ). The most prevalent protocol for frozen-thawed\nembryo transfer is the natural cycle or endometrial preparation with exogenous estrogen and progesterone,\nwith or without the addition of a GnRH\nagonist ( 10 - 12 ).\nBecause the natural cycle protocol does not require\nexogenous hormones‚ it is favored by many\npatients ( 13 ). It has been observed that temporal\ncharacteristics of the endometrium such as the\nformation of pinopodes (markers of endometrial\nreceptivity) are out-of-phase according to measurements\nin normal females who have been placed\non exogenous steroids ( 14 ). Thus, the transfer of\nfrozen-thawed embryos in natural cycles is a favored\noption for women with normal ovulatory\nmenstrual cycles ( 15 ).\nThere is an idea that the endogenous production\nof progesterone is enough to support\nimplantation in a natural cycle. However, an\ninadequate progesterone level at the time of\nimplantation or during early pregnancy may\nhappen naturally due to luteal phase deficiency\n(LPD), which can result in infertility or abortion\n( 16 ).\nThe reported frequency of LPD ranges from\n3.7% to 20% among infertile patients ( 17 ,  18 ).\nThe frequency has been demonstrated to be approximately\n8.1% in natural cycles in normoovulatory\npatients with primary or secondary\ninfertility ( 19 ). Thus, women who undergo frozen-\nthawed embryo transfers may have sub optimal\nendometrium during their natural cycles.\nThere is limited information about the effect\nof luteal phase supplementation on pregnancy\nrate in natural frozen-thawed embryo transfer\ncycles. Therefore, we have designed a prospective\nrandomized study to verify if pregnancy\nrates could be enhanced with progesterone supplementation\nduring the luteal phase and early\npregnancy following a frozen-thawed embryo\ntransfer in a natural cycle.\n\nThe study was designed as a prospective randomized\nclinical trial. A total of 102 women\neach underwent an embryo transfer in a natural\ncycle in Yazd Research and Clinical Center for\nInfertility affiliated by Shahid Sadoughi University\nof Medical Sciences, from March 2011\nto March 2012. This study was approved by the\nEthics Committee of Yazd Research and Clinical\nCenter for Infertility. Prior to starting the\nstudy‚ an informed consent was signed by each\ncouple. The inclusion criteria were: cryop reserved\nembryos after conventional  in vitro  fertilization\n(IVF) or intracytoplasmic sperm injection\n(ICSI)‚ maternal age of 20-40 years (on\nthe day of embryo freezing)‚ regular menstrual\ncycle of 25-35 days, and body mass index of\n20-27 kg/m 2 . Exclusion criteria were: the use\nof testicular sperm for ICSI (ejaculated sperm\nonly)‚ basal follicle stimulating hormone (FSH)\nlevels ≥12 IU/l, stage III-IV endometriosis, and\npolycystic ovarian syndrome (PCOS).\nPatients were randomized to either group in\na ratio of 1:1 by means of computer-generated\nrandom numbers on the day of participation.\nGroup selection and randomization were performed\nby a nurse not involved in the study, by\nusing opaque sealed envelopes. Both the patients\nand the clinicians were aware of the allocated\narm.\nOf the initial 109 women invited to participate,\n102 were included in the study. All women\nhad previously undergone IVF or ICSI with\nembryo cryopreservation. They were randomly\nallocated to either the progesterone (n=51) or\nthe no-progesterone (n=51) groups. In the progesterone\ngroup, we excluded four women. One\npatient had an endometrial polyp and three patients\nhad thin endometria. Similarly, three patients\nwere excluded from the no-progesterone\ngroup because of endometrial polyps ( 2 ) and\none patient who did not return to the study.\nThus, in this study, 102 women each underwent\nan embryo transfer in a natural cycle. The final\nanalysis was performed on 51 patients in each\ngroup. On the second or third days of the menstrual\ncycle, all patients underwent transvaginal\nultrasounds and serum hormone analysis\nfor FSH. Then, a vaginal ultrasonographic examination\nwas performed on cycle days 10 and repeated as necessary. Final oocyte maturation\nwas achieved by intramuscular (IM) administration\nof 10000 IU of hCG (Pregnyl, Daropakhsh,\nIran) when an endometrial thickness of 8\nmm or more and a follicle of 18 mm were present\non the ultrasound. On the day of the hCG\nadministration, we measured serum estradiol‚\nprogesterone and LH levels.\nThe progesterone group received 100mg/day\nof progesterone (Aburaihan Pharmaceutical\nCo., Tehran, Iran) IM, that began 36 hours after\nthe hCG administration and continued until\nten weeks of gestation if pregnancy occurred.\nControl patients received no progesterone. In\nboth groups, cryopreserved embryo transfer\nwas performed with a Cook catheter (Cook Ireland\nLtd.) five days after hCG administration.\nSerum β-hCG level was measured 14 days after\nthe transfer.\nMorphology of fresh cleavage-stage embryos\nwas evaluated according to the number of blastomeres\nand degree of fragmentation. Embryo\nselection for transfer or freezing was performed\nin the morning of the transfer day. Embryos\nwere considered suitable for freezing if they\nhad <30% fragmentation. Cryopreservation of\nall embryos was undertaken with vitrification\nby the cryotop method on day 3 of pre implantation\ndevelopment in both groups. After two-step\nloading with equilibration solution that contained\nethylene glycol and dimethyl sulfoxide\nand a vitrification solution that contained ethylene\nglycol, dimethyl sulfoxide and sucrose,\na narrow glass capillary was used to load the\nembryos onto the cryotop. After loading, the\nmajority of the solution was removed to leave\nonly a thin layer that covered the embryos, after\nwhich the sample was quickly immersed into\nliquid nitrogen. Subsequently, the plastic cap\nwas pulled over the film part of the cryotop and\nthe sample stored in liquid nitrogen. At warming,\nthe protective cap was removed from the\ncryotop while it was still submerged in liquid\nnitrogen and the cryotop was immersed directly\ninto a 37˚C medium that contained sucrose.\nNext, the embryos were sequentially incubated\nin diluent solution before further  in vitro  culture\nfor transfer. Each embryo was carefully evaluated\nimmediately after thawing for the number\nof surviving blastomeres, followed by a second\nevaluation the next morning. Embryos were accepted\nfor transfer if they retained ≥50% of intact\nblastomeres after thawing.\nThe main outcome measures concerned clinical\npregnancy and implantation rates. Chemical\npregnancy was defined as serum β-hCG>50\nIU/L at 14 days after the embryo transfer. Clinical\npregnancy was defined as the presence of\na gestational sac with heart beat identified by\nultrasound 4-5 weeks after the embryo transfer.\nImplantation rate was defined as the ratio of\ngestational sacs to the number of embryos transferred.\nClinical abortion rate was determined as\nclinically recognized pregnancy losses before\n20 weeks of gestation.\nThe SPSS 19 package program was used to\nperform all statistical analyses. The normality of\ndistribution of variables was tested by the Kolmogorov-\nSmirnov test. Independent sample t test\nwas used for continuous variables which were\nnormally distributed and Mann-Whitney U test for\ndata not normally distributed. Chi-square or Fisher\nexact tests were used for qualitative variables as\nappropriate. A p value <0.05 was considered statistically\nsignificant. The data are presented as the\nmean ± standard deviation unless otherwise indicated.\n\nThere were no significant differences noted in the fertilization rate between study and control\ngroups (55.4% vs. 64.3%; p=0.16). Of the 102 patients\nincluded in this study, 51 received progesterone\nand the other 51 did not. Table 1 describes\nthe basic characteristics of the patients in the two\ngroups. The demographic parameters were similar\nin both groups in terms of age, basal FSH levels‚\nbody mass index (BMI)‚ the number of previous\ncycles‚ etiology of infertility, and infertility duration.\nTable 2 compares the previous fresh cycle\ncharacteristics in the two groups.\nThe mean number of oocytes retrieved‚ mean\nnumber of mature oocytes and the number of embryos\nobtained and vitrified did not differ between\nthe groups. There were no significant differences\nnoted in the fertilization rate (55.4% vs. 64.3%;\np=0.16). In addition, the previous stimulation protocols\nand fertilization procedures were similar in\nthe two groups. Only ejaculated sperms had used\nfor conventional IVF or intracytoplasmic sperm\ninjection and percent of sperms with progressive\nmotility and sperms with normal morphology‚ also\nsperm count were not different in those groups.\nThere was no significant difference observed\nbetween the groups regarding the reasons for\nembryo freezing. Table 3 compares the cycle\ncharacteristics of the two groups. Endometrial\nthickness and estradiol‚ progesterone and LH\nlevels on the day of hCG administration were\nsimilar between groups.\nThe cycle length until the day of hCG administration‚\nnumber of embryos transferred, and the\nnumber of good-quality embryos did not differ\nin the two groups. Table 4 presents a comparison\nof the pregnancy outcomes of the study groups.\nAgain, no statistically significant differences were\nobserved in the clinical pregnancy rate between\nthe groups (33.3% vs.27.5%, p=0.66). Although\nthere was a trend toward an increased clinical\npregnancy rate with luteal supplementation‚ the\ndifference was not significant. There were no differences\nbetween the implantation rates (16.6% vs.\n15.3%‚ p=0.93) or clinical abortion rates (11.8%\nvs.14.3%‚ p=0.83). The flowchart of the study is\nshown in figure 1.\nCharacteristics of patients\nPatients’ previous fresh cycle characteristics\nFrozen-thawed embryo replacement cycle characteristics\nPregnancy outcomes\nFlowchart of study patients\n\nThe granulosa cells of the developing follicle generate\nestradiol in response to gonadotropin stimulation\nin natural cycles. The endometrium acquires\nreceptivity to embryo implantation by responding\nto progesterone action on an appropriately primed\nendometrium. Estrogenic stimulation would result in\nendometrial proliferation and the induction of progesterone\nreceptors. The endometrium undertakes\nprofound conformational and biochemical changes,\nfrom proliferative to secretory, with a concomitant\ninduction of endometrial receptivity and opening of\nthe window of implantation in response to progesterone\n( 20 ). During the implantation window, the endometrium\nwhich is unexpectedly unreceptive towards\nembryo implantation acquires a functional condition\nuseful to blastocyst reception ( 21 ).\nThe transfer of frozen-thawed embryos has important\nimplications for the management of women undergoing\novarian hyperstimulation for IVF ( 2 ). Frozen embryo\ntransfer is reported to be successful during the natural\ncycle after spontaneous ovulation according to the literature\n( 22 ). In a study by Morozov et al. a higher pregnancy\nrate was observed in recipients who underwent\nnatural cryothaw cycles than in hormone replacement\ntreatment cycles. In their study the level of estradiol was\ngreater in the substitution cycles when compared with\nthe natural cycle. Regarding those results, we have supported\nthe theory that the window of uterine receptivity\ncloses earlier at a higher endogenous estrogen level and\nlimits the time for the transferred embryos to implant\nsuccessfully ( 9 ). According to their results,hormone\nreplacement treatment versus the natural cycle for cryothaw\nembryo transfer was associated with decreased\npregnancy rates. In the current study, we have evaluated\nthe outcome of hCG-induced natural cryothawed\nembryo transfer cycles that were supported during the\nluteal phase with IM progesterone. We compared this\nwith the outcome of hCG-induced natural cycles in the\nabsence of luteal phase support.\nOur hypothesis was that progesterone support has a\nbeneficial effect on pregnancy rate after frozen embryo\ntransfer in natural cycles, but the results did not support\nour hypothesis. In our study, hCG was used for final oocyte\nmaturation. It was suggested that hCG administered\nfor the final oocyte maturation in stimulated IVF cycles\nwould cause a luteal phase defect by suppressing LH\nproduction through a short-loop feedback mechanism\n( 23 ) although the use of hCG did not down-regulate LH\nsecretion in the luteal phase of regular and unstimulated\ncycles in women with normal ovulation ( 24 ). Additionally,\nin our study none of the patients developed premature\nluteinization. Premature LH surge is defined as\nan LH level of ≥10 IU/L and a progesterone level of\n≥1.0 ng/ml on the day of hCG administration ( 25 ). An\nelevated progesterone level advances the endometrium‚\ntherefore the replacement of day 3 embryos occur in an\nasynchronous endometrium with subsequent failure of\nestablishing an embryo-endometrium cross-dialog, resulting\nin implantation failure ( 26 ).\nBourgain et al. have reported that progesterone induces\na secretory transformation of the endometrium in\nthe luteal phase ( 27 ) and by inducing this change after\nsufficient estrogen priming, progesterone improves\nendometrial receptivity ( 28 ). Progesterone not only\nsupports endometrial development but also maintains\nembryo survival by shifting the immune system toward\nthe production of non-inflammatory Th2 cytokines ( 29 ,\n 30 ). In addition, by inducing nitric oxide synthesis in the\ndeciduas‚ they intensify local vasodilatation and uterine\nrepose ( 31 ). A study by Orvieto et al. has shown that,\nin artificial cryothawed embryo transfer cycles, a highdose\nprogesterone supplementation in the luteal phase\nresulted in a higher clinical pregnancy rate ( 32 ).\nIn contrast to our study, Bjuresten et al. have reported\nthat progesterone supplementation improved the live\nbirth rate after embryo transfer in natural cycles ( 15 ). In\ntheir study, women received vaginal progesterone at a\ndose of 400 mg twice a day from the day of the embryo\ntransfer. They attributed the increase in live birth rate\nto the effects of vaginal progesterone. Vaginal progesterone\nresults in adequate endometrial development, in\nspite of low serum progesterone levels.\nOur study was in agreement with a study by Kyrou et\nal. that reported luteal phase support did not affect ongoing\npregnancy rates in natural hCG-induced frozenthawed\nembryo transfer cycles ( 33 ). A possible reason\nfor our finding was that the women in the present study\nhad a normal ovulatory function; those with ovulatory\ndysfunction were excluded from the study. Luteal phase\ndefect in stimulated IVF cycles is due to supra physiological\nlevels of steroids which directly inhibit the LH\nrelease via negative feedback actions at the hypothalamic-\npituitary axis level ( 34 ). However it seems that LPD\nis not a main etiologic factor for implantation failure in\nnatural frozen thawed embryo transfer cycles.\n\nThere emerged no significant differences between the two groups in our study with regards to the implantation\nor clinical pregnancy rates‚ but there was\na trend toward an increased clinical pregnancy rate\nwith luteal supplementation. Thus, further studies are\nneeded to confirm our findings.","source_license":"CC-BY-4.0","license_restricted":false}