Clinical Observation And Mechanism Study Of Luteal Phase Deficiency In Patients With Diminished Ovarian Reserve

ObjectivesDiminished ovarian reserve(DOR)refers to a decline in female fertility,defined as a decrease in the number of ovarian oocytes accompanied by changes in ovarian reserve indicators,which may exacerbate infertility management difficulties in women.DOR is a common cause of female infertility and is one of the most difficult diseases to treat clinically.Previous studies have found that DOR women have an increased risk of recurrent abortion and complications during pregnancy.Adverse pregnancy outcome is associated with luteal phase deficiency(LPD),but the clinical evidence is not sufficient at present.LPD is associated with various factors,such as follicular dysplasia and decreased estrogen secretion;Low LH pulse peak and insufficient progesterone secretion,among which the functional defects of granulosa cells lead to poor luteinization of granulosa cells after ovulation is an important cause of LPD.Our previous studies found that autophagy defects can cause granulosa cell differentiation disorder and affect follicular development,but the role of autophagy defects in the occurrence of LPD are still unclear.Based on the above background,on the one hand,our study determined whether DOR patients had luteal phase deficiency and the related risks of maternal and infant complications in pregnancy and perinatal period by using the clinical data of DOR women during the process of ART and follow-up information of pregnancy and perinatal complications.On the other hand,we explored the role and mechanism of granulosa cell autophagy defect in the occurrence of LPD in DOR women,providing theoretical basis for the occurrence mechanism of LPD in DOR patients and new ideas for clinical LPD intervention.Methods(1)Clinical study on the association between DOR and LPD:Patients under 40 years old who underwent the first cycle of in vitro fertilization/intracytoplasmic sperm injectionembryo transfer(IVF/ICSI-ET)in Reproductive Hospital affiliated to Shandong University from January 2014 to December 2014 were enrolled and were divided into DOR group and control group according to ovarian reserve index.Progesterone levels of both groups were collected before IVF/ICSI-ET.DOR group:Basic serum follicle stimulating hormone(FSH)level≥10IU/L,anti-Müllerian hormone(AMH)<1.2 ng/mL;Control group:FSH<10 IU/L,AMH≥1.2 ng/mL.The control group was matched with the DOR group by age and body mass index(BMI).Progesterone level in the middle luteal phase(6-8 days after ovulation)were measured according to the time of ovulation.Mid-luteal progesterone levels in DOR group and control group were compared to evaluate the difference in luteal function between the two groups.(2)Clinical study of maternal and infant complications in DOR patients:Patients under 40 years old who underwent IVF/ICSI-ET in Reproductive Hospital affiliated to Shandong University from January 2012 to June 2019 and received single fetal live birth were enrolled and divided into DOR group and control group according to ovarian reserve indicators.The inclusion criteria are the same as in part I.The control group was matched with the DOR group by age and BMI.The risk of common maternal and infant complications was compared between the two groups,including hypertensive disorders of pregnancy(HDP),preterm birth(PTB),low birthweight,Gestational diabetes Mellitus(GDM)and placenta previa.(3)The role and mechanism of autophagy defects in the process of luteinization in granulosa cells:The effect of autophagy defect on the luteinization of granulosa cells and its related mechanism were investigated by in vivo and in vitro functional experiments.Human granulosa cells SVOG and KGN were used to induce autophagy defects by siRNA interference of ATG5 and BECN1,or by using chloroquine,an autophagy inhibitor.Then,progesterone levels in cell culture medium,mRNA and protein expression levels of StAR and CYP11A1,and protein expression levels of LHR were detected.hCG was further used to activate LH signaling pathway in granulosa cells,and the phosphorylation levels of ERK1/2 and CEBPβ were detected by Western blot to determine the effect of autophagy defect on LH signaling pathway in granulosa cells.Meanwhile,mRNA and protein expression levels of Relaxin2,a factor associated with gestational hypertension,were detected.In vivo experiments,we induced autophagy defects in mice by intraperitoneal injection of the chloroquine,then detected the serum progesterone level,observed the morphological changes of ovarian tissues,and detected the expression of StAR and CYP11A1.Results(1)Clinical study on the association between DOR and LPD:By comparing the progesterone levels of 160 DOR patients and 285 control female,we found that the serum progesterone levels of DOR patients in mid-luteal period were significantly lower than those of control group(11.18±5.40 ng/mL vs.13.09±5.99 ng/ml,P=0.002).These results suggest that DOR patients have an increased risk of LPD.(2)Clinical study of maternal and infant complications in DOR patients:We followed up 193 DOR patients and 386 control female for maternal and infant complications.The risk of hypertensive disease of pregnancy was significantly increased in the DOR group compared with the control group(5.7%vs.2.1%,P=0.021).There was no significant difference between the two groups in the incidence of gestational diabetes mellitus(5.7%vs.5.2%,P=0.794),placenta previa(1.6%vs.1.8%,P=1.000),preterm birth(10.9%vs.7.5%,P=0.174),low birthweight(6.2%vs.5.4%,P=0.704).(3)Effect and mechanism of autophagy defect on luteinization of granulosa cells:Inhibition of autophagy in SVOG and KGN cell lines reduced progesterone synthesis,mRNA and protein expression of StAR,CYP11A1 and Relaxin2,and protein expression of LHR.Meanwhile,the expression of p-ERK and p-CEBP β induced by hCG was significantly decreased,suggesting that autophagy defects lead to the activation disorder of LH signaling pathway in granulosa cells.After inhibition of autophagy in mice,serum progesterone level was significantly reduced,luteal formation was abnormal,and the protein expression of StAR and CYP11A1 in ovary were significantly decreased.Conclusion(1)Compared with women with normal ovarian reserve,the level of mid-luteal progesterone in DOR patients was significantly reduced,suggesting that the patients with DOR may have increased risk of luteal phase deficiency.(2)Compared with women with normal ovarian reserve,DOR patients have an increased risk of hypertensive disorders of pregnancy.It is recommended to pay attention to the changes of blood pressure in DOR patients during pregnancy and strengthen pregnancy and perinatal monitoring.(3)Autophagy defects in granulosa cells lead to decreased expression of LHR,impaired activation of LH pathway,and decreased expression of StAR and CYP11A1,ultimately leading to inadequate progesterone synthesis.In addition,autophagy defects also resulted in decreased Relaxin2 expression in granulosa cells,which may be related to hypertension during pregnancy.This study provided a new target for clinical intervention of LPD in DOR patients.