Abstract： Objective:To investigate the effects of supplementation of recombinant luteinized hormone (rLH) and its timing on pregnancy outcomes of patients at 35 years or older with follicular-phase long protocol.Methods:Clinical data of women undergoing in vitro fertilization or intracytoplasmic sperm injection with follicular-phase long protocol was collected and retrospectively analyzed in the First Affiliated Hospital of Nanjing Medical University from January 2017 to December 2019. There were 558 patients at 35 years or older included in this study, and they were divided into three groups: group A was patients with only recombinant follicle stimulating hormone (rFSH) stimulation (127 cycles), group B was patients with rFSH plus rLH supplementation in the mid-follicular phase (141 cycles), and patients in group C received combined rFSH and rLH from the first day of ovarian stimulation (290 cycles). The basic characteristics of patients of each group were observed and the effects of ovarian simulation and pregnancy outcomes were compared among the three groups. Logistic regression model was performed to explore the association between different groups and pregnancy outcomes.Results:The basic characteristics such as age, duration of infertility, body mass index (BMI) and serum basic follicle stimulating hormone (FSH) were comparable among the three groups (all P>0.05). Anti-Müllerian hormone (AMH), antral follicles count (AFC) and basic luteinized hormone (LH) were significantly lower in group C compared to group A and group B (all P<0.05). There were statistically significant differences in initiation dosage, total dosage and duration of gonadotropin (Gn) among the three groups (all P<0.01), the initiation dosage, total dosage and duration of Gn were higher in group C than the other two groups. The number of oocytes retrieved and available embryos were significantly lower in group B and group C than group A (all P<0.001). In fresh embryo transfer cycles, significantly higher implantation rate (45.3%, 117/258) and clinical pregnancy rate (52.6%, 111/211) were found for group C when compared with group A and group B ( P=0.036, P=0.006). The live birth rate in fresh embryo transfer cycles was comparable among the three groups ( P=0.098). The implantation rate, clinical pregnancy rate and live birth rate in the subsequent frozen-thawed embryo transfer cycles did not differ significantly among the three groups (all P>0.05). There were no significantly differences in the cumulative pregnancy rate and the cumulative live birth rate among the three groups (all P>0.05). After adjusted for age, BMI, AMH, AFC, basic FSH and LH, total Gn dosage, endometrial thickness at transfer, number of oocytes retrieved, number of embryos transferred and stage of embryo transferred, in fresh embryo transfer cycles, the clinical pregnancy rate (adjusted OR=2.793, 95% CI: 1.512-5.162, P<0.001) and live birth rate (adjusted OR=2.324, 95% CI: 1.241-4.351, P=0.008) were higher in group C, while clinical pregnancy rate and live birth rate were similar between group B and group A in fresh embryo transfer cycles (all P>0.05); there was no significant difference in cumulative live birth rate among the three groups ( P>0.05). Conclusions:The supplementation of rLH from the first day of ovarian stimulation improves the pregnancy outcomes of patients at 35 years or older in fresh embryo transfer cycles during follicular-phase long protocol. However, the supplementation of rLH has no benefit on cumulative live birth rate.