The Preliminary Study About The Roles And Mechanisms Of Beta2-glycoprotein Ⅰ In Reproduction

Part OneObjectives:To confirm the existence of β2GP I in normal female mature follicular fluid and determine its concentration level; to observe the expression levels and expression differences of β2GP I in follicular fluid of normal control, PCOS and decreased ovarian reserve patients, the expression levels and expression differences of β2GP I in normal control pregnancy group and normal control non-pregnancy group, normal control different age subgroups; to analyse the correlation between β2GP I concentration levels and serum estradiol (E2) levels of hCG day, number of oocytes and oocyte maturation rate in IVF/ICSI cycles, and to explore the effect of β2GP I on proliferation and apoptosis of ovarian granulosa cells.Methods:Selected100female patients for in vitro fertilization and embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) due to the simple tubal factor or male factor as normal control group, then divided into pregnancy group (n=58) and non-pregnancy group (n=42) according to pregnancy outcome, divided into≤25years old group (n=16),26-30years old group (n=35),31-35years old group (n=30), and>35years old group (n=19) according to age;85PCOS patients and62decreased ovarian reserve patients for IVF-ET/ICSI in the corresponding period were also selected. Conventional long protocol for ovulation induction, collected the serum on hCG day and the follicular fluid of dominant follicle on oocyte retrieval day. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of β2GP I in follicular fluid, and electrochemiluminescence immunoassay was used to detect the E2levels in serum.Results:The concentration level of β2GP I in normal female mature follicular fluid is0.53-2.89mg/L, and mostly concentrated in the range of0.8-2.0mg/L. The concentration levels of β2GP I between normal control pregnancy group and normal control non-pregnancy group were not statistically different (1.18±0.34vs.1.25±0.33, P>0.05), neither showed any remarkable difference among≤25years old group,26-30years old group,31-35years old group, and>35years old group (1.16±0.35、1.27±0.32、1.16±0.43、1.09±0.34, P>0.05),26-30years old group was higher and>35years old group was lower. The concentration levels of β2GP I in PCOS group and decreased ovarian reserve group were both higher than the normal control group with no statistically significant difference(1.47±0.31vs.1.21±0.41,1.52±0.38vs.1.21±0.41, P>0.05). The concentration level of β2GP I in normal control group follicular fluid is negatively correlated with the serum estradiol (E2) level of hCG day, number of oocytes and oocyte maturation rate (r=-0.279, P<0.05; r=-0.243, P<0.05; r=-0.711, P<0.01).Conclusions:Normal female mature follicular fluid contains the P2GP I. The concentration level of β2GP I is negative trends related with pregnancy outcomes, and has some relevance with age distribution. The elevated level of β2GP I may indicate pathological conditions. Under normal physiological conditions, P2GP I plays a negative regulatory role in the granulosa cell proliferation, apoptosis, and steroid hormone secretion, then participate in the development of follicles.Part TwoObjectives:To observe the effects of α2GP I that in normal female mature follicular fluid with different concentrations and different exposure time on human sperm motility in vitro.Methods:100low motility semen samples from asthenospermia patients were collected for research, each semen sample was divided into4groups, one as the NS control group and the others incubated in vitro with P2GP I that in normal female mature follicular fluid at three different concentrations1.0mg/L,1.5mg/L and2.0mg/L respectively, under37℃for2h. Semen routine analysis was carried out by computer assisted sperm analysis (CASA) at30min,1h and2h.Results:Compared with the NS control group, β2GP I at1.5mg/L and2.0mg/L significantly increased the sperm motility amplification Δ(a+b)%at30min,1h and2h (P<0.05), β2GP I at1.0mg/L effected no significant difference at30min for Δ (a+b)%(P>0.05), but showed significant difference at1h and2h (P<0.05). There was no significant difference between1.5mg/L β2GP I and1.0mg/L β2GP I at30min for Δ (a+b)%(P>0.05), but1.5mg/L β2GP I significantly increased the Δ (a+b)%than1.0mg/L β2GP I at1h and2h (P<0.05).2.0mg/L β2GP I significantly increased the Δ (a+b)%than1.0mg/L β2GP I at30min,1h and2h (P<0.05); so did the Δ (a+b)%of2.0mg/L β2GP I significantly increased than1.5mg/L β2GP I at30min,1h and2h (P<0.05). Compared with the NS control group,2.0mg/L β2GP I significantly increased the VSL. ALH,LIN、WOB and SRT at30min (P<0.05). Compared with the NS control group,1.5mg/L β2GP I and2.0mg/L β2GP I both significantly increased the VSL、ALH、LIN、 WOB and SRT at1h(P<0.05);2.0mg/L β2GPI significantly increased the VSL. ALH. LIN. WOB and SRT than1.0mg/L β2GP I at1h (P<0.05). Compared with the NS control group,1.5 mg/L (β2GP I and2.0mg/L β2GP I both significantly increased the VSL. ALH、LFN、WOB and SRT at2h (P<0.05);2.0mg/L β2GP I significantly increased the ALH、LPN、WOB and SRT than1.0mg/L β2GP I at2h (P<0.05).Conclusions:The β2GP I in normal female mature follicular fluid is able to improve sperm motility in vitro with concentration-dependent and time effect.Part ThreeObjectives:To observe the effects of recombinant β2GP I (rβ2GPI) with different concentrations on human sperm motility in vitro, and the concentration change of HBsAg after interacting with rβ2GPI. To investigate the value of rβ2GPI in treatment for asthenospermia associated with HB V infection.Methods:80low sperm motility and HBsAg positive semen samples from asthenospermia patients with HBV infection were collected for research. Each semen sample was divided into5groups, one as the HBsAg quantitative control group, one as the NS control group, and the others incubated in vitro with rβ2GP I at three different concentrations5umol/L,10umol/L and20umol/L respectively, under37℃for2h. Semen routine analysis was carried out by computer assisted sperm analysis (CASA), and electrochemiluminescence immunoassay was used to determine the HBsAg concentrations.Results:Compared with the NS control group, rβ2GP I at5umol/L,10umol/L and20umol/L significantly increased the sperm motility (a+b)%(P<0.05); there was no significant difference between10umol/L rβ2GP I and5umol/L rβ2GP I about the (a+b)%(P>0.05); the (a+b)%of rβ2GP I at20umol/L significantly increased than rβ2GP I at5umol/L and10umol/L (P<0.05).10umol/L rβ2GP I and20umol/L rβ2GP I both significantly increased the VSL、ALH、LFN、 WOB and SRT than the NS control group (P<0.05);20umol/L rβ2GP I significantly increased the VSL, ALH、LIN、WOB and SRT than5umol/L rβ2GP I and10umol/L rβ2GP I (P<0.05). Compared with the HBsAg quantitative control group, rβ2GP I at20umol/L significantly decreased the HBsAg concentration after interacting with HBsAg positive semen samples (P<0.05), but neither showed any remarkable effects at5umol/L rβ2GP I and10umol/L rβ2GP I (P>0.05). HBsAg concentrations of HBsAg positive semen samples after interacting with rp2GP I at5umol/L,10umol/L and20umol/L for pairwise comparisons were not statistically different (P>0.05).Conclusions:The recombinant β2GP I (rβ2GPI) is able to improve sperm motility in vitro with concentration-related manner. The rβ2GPI could reduce the concentration of HBsAg by interacting with HBsAg positive semen, and provide a new way of thinking about the therapy for asthenospermia associated with HBV infection.