{"paper_id":"9b685a6b-37d6-46e6-992d-bc6ab38fcf73","body_text":"Multiple studies and meta-analyses have reported\nthe importance of luteinizing hormone\n(LH) or LH mediated activity for  in vitro  fertilization\n(IVF) stimulation cycles ( 1 - 4 ). More precisely,\nadministration of human menopausal gonadotropin\n(hMG) has led to increased pregnancy,\nclinical pregnancy, and live birth rates compared\nto recombinant follicule-stimulating hormone (r-\nFSH) alone ( 1 ,  2 ,  5 ). LH is available in two forms,\nrecombinant (r-LH) or in hMG, which contains\nhuman chorionic gonadotropin (hCG) that acts\nas an LH analogue. Few studies have evaluated\nthe role of different types of LH stimulation by\ncomparing r-LH to HMG, which may yield subtle\ndifferences. A previous study performed at the\nMcGill Reproductive Center compared subjects\nwho received r-LH and r-FSH to subjects that received\nhMG alone in women with good or poor\novarian reserve. In subjects with good ovarian reserve,\nthe r-LH group had higher numbers of oocyte\nand embryos, increased pregnancy rates per\ncycle, and overall higher clinical pregnancy rates\nwhich showed a potential benefit for r-LH stimulation\n( 6 ). However, no distinction was made between\nIVF protocols.\nThe current study compared stimulation parameters,\npregnancy and clinical pregnancy rates of\npatients with normal ovarian reserve parameters\ntreated with a long gonadotropin releasing hormone\n(GnRH) agonist protocol and received r-LH\nto those treated with hMG that contained hCG as\nan LH analogue. Both groups of participants also\nreceived daily FSH stimulation.\n\nWe performed a cohort study from data collected\nat the McGill Reproductive Center. An analysis of\nIVF cycles for a two-year period was undertaken\nto identify all patients treated at our institution\nthat met the inclusion criteria. To be included in\nthe study patients received FSH and either r-LH\nor HMG but not both forms of LH stimulation.\nPatients with maximum serum baseline FSH levels\nunder 10 IU/L (drawn menstrual cycle days 2\nto 5 inclusively) and baseline follicle counts of 6\nfollicles or more determined by transvaginal ultrasound\n(TVUS) as assessed on menstrual cycle days\n2 to 5, inclusively, initiated treatment with a long\nGnRH agonist down-regulation protocol (n=122).\nA total of 65 women received r-LH whereas 57\nreceived hMG. Cycles were excluded from analysis\nif the patient had hyperprolactinemia (morning\nfasting prolactin greater than 26 ng/mL), thyroid\nabnormalities (TSH below 0.39 or above 4.0 μIU/\nmL), hypothalamic pituitary dysfunction, and\novarian failure (FSH below 2 IU/L or abover 20\nIU/L and estradiol <66 pg/mL). The McGill University\nCommittee for the Protection of Human\nResearch Subjects approved this data collection.\nAll subjects were de-identified in the database.\nPatients were allotted to their respective treatment\nregimens by clinic staff to maintain equivalent\nrates for prescription of different drugs produced\nby competing pharmaceutical companies. Patients\nthat received hMG (Repronex, Ferring Canada,\nNorth York, ON) also received either follitropin\nbeta (64%, Merck Canada, Inc., Pointe-Claire,\nQC), follitropin alfa (20%, EMD Serono Canada,\nMississauga, ON), or purified urofollitropin (16%,\nFerring Canada, North York, ON). All patients that\nreceived r-LH were treated with follitropin alfa\n(EMD Serono Canada, Mississauga, ON).\nPatients treated with a GnRH agonist long downregulation\nprotocol initially received stimulation\nwith 112.5 to 225 units of FSH daily at the discretion\nof their treating physician. The physician selected\nthe dose based on parameters of ovarian reserve\nnoted during the planning of the cycle. After\n5 days of FSH stimulation, we reassessed the doses\nwhich were titrated up or down depending on\nserum estradiol levels, as well as the numbers and\ndiameters of follicles noted. Subsequently, patients\nwere monitored at 1 to 3 day intervals with serum\nestradiol levels and transvaginal ultrasonographic\nfollicle monitoring. Patients were prescribed LH\nactivity such that the ratio of FSH to LH was 3:1\nto 2:1 at the discretion of their treating physician.\nWe measured peak serum estradiol levels and\neither 10000 IU or 5000 IU of hCG were given\nbased on our center’s protocol 35 hours prior to\negg retrieval. We followed the McGill Reproductive\nCenter protocol for egg retrieval and embryo\ntransfer using either a Cook (Cook Canada, Inc.,\nStouffville, ON) 17-gauge single lumen needle\nor a 16-gauge double lumen flushing needle and\nwarmed saline flush. Pressure for aspiration was\nkept at 145 mmHg by a Cook Vacuum Pump (KMar\n8200, Cook, Australia).\nAt 17 to 19 hours after insemination, embryo fertilization was evaluated for the presence of two pronuclei and two polar bodies. The zygotes were transferred to an IVF cleavage medium for further culture (Cook, Australia). The embryos were evaluated on days 2 (41-43 hours after insemination) and 3 (65-67 hours after insemination). Quality of development of the embryos was assessed according to the regularity of blastomeres, the percentage and pattern of anucleate fragments, and dysmorphic characteristics. Good quality embryos on day 2 had at least 2 cells and by day 3, they at least 6 cells with less than 20% anucleate fragments and no apparent morphological anomalies. Embryos were considered low quality if they showed blastomere multi-nucleation, poor cell adhesion, uneven cell division, and cytoplasmic anomalies. We transferred embryos of best quality based on cell number, degree of fragmentation, symmetry of blastomeres, degree of compaction, clarity and texture. Depending on age and physician orders, the transfer was performed on day 2 or 3.\nEmbryos were transferred under trans-abdominal ultrasound guidance and placed 2.5 to 1.5 cm from the uterine fundus using a Wallace embryo replacement catheter (Smith Medical International Ltd., UK). The number of embryos transferred varied between 2-5 depending on the patient’s age, embryo quality, and previous number of unsuccessful IVF cycles. Decisions were based according to recommendations by the American Society for Reproductive Medicine Committee Opinion ( 7 ). Progesterone was prescribed for luteal phase support per the center’s protocol. We defined pregnancy as a single serum hCG level of ≥20 IU/L measured 16 days after egg retrieval. Clinical pregnancy was defined as an intra-uterine positive fetal heartbeat seen on TVUS at 6-7 weeks of gestational age.\nStatistical analysis was done using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were evaluated for normal distribution using the Kolmogorov-Smirnov test. All continuous variables were normally distributed. We performed stepwise logistic regression on continuous and categorical variables to control for multiplicity and confounding effects. Rates for causes of infertility were analyzed by chi-squared tests. Data are presented as means ± SD with statistical significance as a two-sided P≤0.05. Controlled variables included patient age, basal serum FSH level, basal serum estradiol level, antral follicle count, previous pregnancies, previous full term pregnancies, previous miscarriages, previous IVF cycles, total LH and FSH stimulation doses, days of LH and FHS stimulation, as well as the number of oocytes and embryos obtained.\nThere were no cancelled cycles in any of the patients. Patients were good responders; hence, we did not anticipate any poor response. Ovarian hyperstimulation syndrome was avoided in all cases. No statistical differences in pregnancy rate (P=0.432) or clinical pregnancy rate (P=0.381) existed among the cycles that used follitropin alfa, follitropin beta or urofollitropin, which was combined in all cases with hMG. This result supported the combined analysis of these results. A comparison of patient demographics in subjects treated with the long GnRH agonist protocol is presented in Table 1. The two groups did not differ in any of the baseline characteristics studied, including basal serum FSH level, basal serum estradiol level, and baseline follicle count. The number of embryos transferred did not differ between r-LH (2.2 ± 0.6) versus hMG (2.3 ± 0.6, P=0.36).\nPatient demographics (mean ± SD)\nAnalysis performed with stepwise logistic regression.\nr-LH; Recombinant human luteinizing hormone, hMG; Human menopausal gonadotropin, IVF;  In vitro  fertilization, and FSH; Follicle stimulating hormone.\nThere were similar rates for causes of infertility in subjects treated with the long GnRH agonist protocol that received either r-LH or hMG (P=0.469).\nA comparison of the r-LH or hMG groups showed that the rates of male factor infertility were 54% (r-LH) and 39% (hMG), the unexplained infertility rates were 32% (r-LH) and 33% (hMG), the rates of endometriosis were 4% (r-LH) and 12% (hMG), and the anovulation rates were 2% in both groups. Tubal factor infertility did not occur in either group.\nTable 2 shows treatment outcomes for the r-LH and hMG groups when treated with the long GnRH antagonist protocol. Patients treated with r-LH had a higher pregnancy rate per cycle start (P=0.0250) and implantation rate (P=0.001) after controlling for patient age, baseline FSH and estradiol levels, antral follicle count, previous pregnancies, full term deliveries and spontaneous abortions, number of previous IVF cycles, dose of FSH and LH administered, as well as days of stimulation. Patients treated with r-LH had a trend towards a higher clinical pregnancy rate per cycle start (P=0.0649). Patients that received r-LH compared to using hMG had more oocytes collected and more embryos created, even though the r-LH group used lower doses of FSH and LH. The number of days of stimulation did not differ between the r-LH and hMG groups.\nIVF cycle characteristics and treatment outcomes\nAnalysis performed with stepwise logistic regression.\nIVF;  In vitro  fertilization, r-LH; Recombinant human luteinizing hormone, hMG; Human menopausal gonadotropin, FSH; Follicle stimulating hormone, LH; Luteinizing hormone, and ICSI; Intracytoplasmic sperm injection.\n\nIn this study, there were lower FSH and LH doses required for stimulation in the long GnRH stimulation cycle with r-LH compared to hMG. r-LH treated subjects had larger numbers of oocytes and embryos obtained compared to hMG treated subjects. Pregnancy rates per cycle start and implantation rates were higher for patients in the r-LH group compared to the hMG group. There was a trend for increased clinical pregnancy rate in the r-LH group; however, this did not reach statistical significance when controlling for confounders. We observed these findings even after controlling for patient age, baseline FSH and estradiol levels, antral follicle count, previous pregnancies, full term deliveries, spontaneous abortions, and previous number of IVF cycles.\nA systematic review and meta-analysis by Coomarasamy et al. ( 3 ) determined that the use of gonadotropins with LH as well as with FSH activity delivered as urinary hMG was shown to be superior to the use of r-FSH alone in long GnRH down-regulation protocols. They showed that the use of hMG was associated with a 4% increase in live birth rates compared to r-FSH alone. Other potential benefits to LH activity might also exist. Weghofer et al. ( 8 ) compared patients who underwent long protocol stimulation with either r-FSH or hMG. They found an improvement of embryonic ploidy in patients stimulated by hMG. However, the importance of the source of that LH activity should be further investigated. LH mediated activity can be administered in two forms, hMG and r-LH. LH activity in hMG is primarily achieved through hCG that acts as an LH analogue. There exist theoretical problems with hMG. For example, the risk of injection of prions through this urinary derived product, which may discourage patients and physicians from its use ( 9 ). As well,  in vitro  studies have demonstrated that r-LH and hCG result in different gene activation of the ovarian cumulous cells and endometrium ( 10 ). Therefore, r-LH may confer different beneficial effects than hMG. This difference in the endometrium may partially explain the increase in implantation seen with embryos achieved after r-LH as opposed to hMG treated cycles.\nTo date, few  in vivo  studies have been published. Hence, it is unclear which group of patients would benefit most from r-LH. Moro et al. ( 11 ), in a randomized controlled trial that enrolled patients over 35 years of age, found no benefit to r-LH over highly purified hMG. A study conducted in the McGill Reproductive Center observed no benefits between subjects with extremely poor ovarian reserve (baseline follicle counts less than 6) who received r-LH and r-FSH compared to subjects who received hMG. However, r-LH was found to be advantageous in terms of pregnancy and clinical pregnancy rates compared to hMG in patients with good ovarian reserve. No distinction was made between the different IVF protocols used (long agonist versus microdose flair) ( 6 ). A study by Requena et al. ( 12 ) compared endocrine profiles of 50 oocyte donors that received either r-LH plus r-FSH together or hMG and urinary FSH. Although there were more oocytes retrieved in the r-LH plus r-FSH group, a lower proportion were in metaphase II. Serum steroid levels did not differ on the day of triggering. In recipients, the implantation and ongoing pregnancy rates were the same in both groups (46.1%). However, as the recipients were not subject to LH stimulation, the difference observed between these results and the current study could be related to the possibility that LH might have a beneficial effect at the level of the endometrium as well. Conversely, a multicenter randomized controlled trial performed in Italy assessed outcomes for two groups of patients who underwent IVF using a down-regulation protocol. The first group received r-FSH plus r-LH, whereas the second group only received urinary hMG. Both groups had the same pregnancy and implantation rates. A lower cost for the IVF cycle was noted in the hMG group, as they used less FSH ( 13 ).\nOur data suggests that r-LH might be beneficial compared to hCG in terms of LH mediated activity in long GnRH agonist cycles. This was a retrospective study, hence, further studies should be undertaken to confirm these results. It would have been interesting to compare follitropin-alpha and r-LH versus hMG and follitropin-alpha. The number of patients treated with this protocol was too small for comparison and should be reassessed in future studies.\n\nFSH plus r-LH may offer benefit compared to FSH plus hMG for ovarian stimulation in long GnRH-agonist protocols performed in good responders. This may occur through different stimulation of the ovarian cumulous cells or endometrium. Further studies, both larger and prospective, are needed to confirm these results.","source_license":"CC-BY-4.0","license_restricted":false}