| Background Vitamin D is a steroid hormone produced by the skin after exposure to ultraviolet light.In addition to regulating the balance between calcium and phosphorus for bone health,studies showed that vitamin D receptors exist in uterus,ovaries,testes and other reproductive organs,which are important for the growth and development of endometrium and follicles,and the quality of sperm.Abnormal expression of vitamin D metabolic pathway genes,such as CYP2R1,CYP27B1,CYP24A1,GC and vitamin D receptor?VDR?,may affect the level of vitamin D,thereby affecting the function of reproductive function.In 1978,the first test-tube baby was born in the world,and then the Assisted Reproduction Technique?ART?develop rapidly.With the incidence of infertility increasing,ART has been known and accepted by most people.However,the success rate of ART reaches a bottleneck,at a level of 50%.Many studies have showed that the level of vitamin D affects the rates of clinical pregnancy and live birth,but the results are controversial.Researches about the distribution and influencing factors of vitamin D based on large data is absent in infertile people.What's more,whether the vitamin D has specific effect on ART is still unknown.As one of the important factors affecting clinical pregnancy rate,embryo quality can be predicted and improved through the study on the relationship between the level of vitamin D and egg obtain rate,normal fertilization rate and high quality embryo rate in ART.CYP2R1,CYP27B1,CYP24A1,GC and VDR,as the key regulatory genes,play an important role in vitamin D metabolism and transport,and are closely related to many important physiological functions of follicle production,placental hormone secretion and endometrial inflammatory factor production.Its abnormal expression will affect the level of vitamin D and its biological function in body.This study will adopt a progressive and in-depth way,from the distribution and related factors of vitamin D in infertility population,to the relationship between vitamin D and fertilization,embryo quality in assisted reproductive technology,and finally explore the expression of vitamin D metabolic pathway-related genes,in order to provide clues for improving the clinical pregnancy rate in infertility population.Part ? Analysis of the levels and related factors of vitamin D in infertility populationObjectives To study the distribution of vitamin D level in the infertile population and analyze the relationship between the levels of vitamin D and the basic conditions of infertile patients and related laboratory indicators.Materials and Methods1.Subjects?1?This is a retrospective study,including 12,261 infertility patients,who accepted ART for the first time in Reproductive Medicine Center at the third Affiliated Hospital of Zhengzhou University from June 2016 to December 2018.?2?Inclusion criteria: In Vitro Fertilization?IVF?,Intracytoplasmic Sperm Injection?ICSI?,Preimplantation Genetic Diagnosis?PGD?,(Preimplantation Genetic Diagnosis?PGS?,Artificial Insemination by Husband?AIH?,Artificial Insemination by donor?AID?was performed for specific reasons,including fallopian tube factors,ovulation disorders,endometriosis,premature ovarian failure,recurrent abortion,male factors?severe hypospermia,asthenospermia?and other unknown reasons.?3?Exclusion criteria: patients with contraindications to assisted reproductive technology;have a history of taking vitamin D supplements within half a year;serious medical diseases such as hypertension and diabetes.?4?Groups: According to the levels of serum 25?OH?D,the patients were divided into two groups: vitamin D deficiency group?25?OH?D? 50 nmol/L?and vitamin D non-deficiency group?25?OH?D > 50 nmol/L?.2.Methods?1?Record the demographic data of the patients,including age,infertility years,BMI,blood pressure,family history;laboratory indicators,such as blood type,liver and kidney functions,the levels of insulin,serum 25?OH?D and basal endocrine,seasons,PCOS,endometriosis,etc.?2?Statistical method:All data is analyzed by SPSS21.0 statistical software,and is expressed by mean ± standard deviation.The categorical variables are compared by chi-square test between groups.If more than 20% expected values are less than 5,Fisher's exact test is used.The continuous variables are analyzed by Student's t test or Mann-Whitney test according to whether they obey normality and homogeneity of variance,and multivariate analysis is performed by two-class logistic regression.Difference of P < 0.05 was considered significant for the statistical tests.Results1.Comparison of demographic data of the study subjects.There was no significant difference in BMI,blood type,family history and endometriosis between the VD-deficiency group and the non-deficiency group in female patients?P>0.05?.2.The proportion of groups according to the levels of vitamin D.There were 10077 cases with VD deficiency,accounting for 82.19%,and 2184 control cases,accounting for 17.81%.3.There was significant difference in age,determination season,insulin resistance and PCOS between two groups?P<0.05?.4.Regression analysis after adjusting the confounding factors.?1?BMI,PCOS,blood type,family history,endometriosis did not affect the levels of vitamin D?P>0.05?.?2?Older age?>35 years old?is a protective factor for vitamin D?P<0.05?.Women ?35 years old are more tend to vitamin D deficiency than women >35 years old.?P<0.05?.?3?Season affects the levels of vitamin D,which was higher in summer and autumn than in winter and spring?P<0.05?,and was significantly higher in spring than in winter?P<0.05?.?4?Insulin resistance affects vitamin D level: Insulin resistance is more prone to vitamin D deficiency than non-insulin resistance group.?P<0.05?.Brief Summary Vitamin D deficiency is common in infertility patients.Age,season and insulin resistance are related to the levels of vitamin D.BMI,PCOS,blood type,family history,endometriosis are not related to the levels of vitamin D.Part ? The relationship between the levels of vitamin D and Oocyte and the embryo quality in ARTObjectives To explore the relationship between the serum and the follicular fluid levels of vitamin D,and to study the role of the follicular fluid levels of vitamin D on oocyte rate,normal fertilization rate,high-qualified embryo rate.Materials and Methods Subjects?1?From December 2018 to January 2019,196 patients were selected as subjects,who received IVF in our hospital.?2?The inclusion criteria were as follows: female age?35years,male age<40years,anti-Mullerian hormone?AMH?>1.1ng/ml or the number of basal sinus follicles>7,all adopting long-term regimen?including long-term luteal phase and long-term regimen?.?3?Exclusion criteria were follows:?i?serious internal diseases;?ii?combined with other endocrine diseases affecting metabolism?such as thyroid dysfunction,hyperprolactinemia,etc.?;?iii?endometriosis;?iv?have a history of taking vitamin D supplements within half a year.?4?Groups: According to the levels of vitamin D in follicular fluid,patients were divided into three groups: vitamin D sufficiency group?34 cases??25?OH?D>75nmol/L?,vitamin D insufficiency group?64 cases??25?OH?D 50-75nmol/L?,vitamin D deficiency group?98 cases??25?OH?D < 50nmol/L?.Methods?1?Record the demographic data of the patients,basic insulin,serum and follicular fluid levels of 25?OH?D,and the levels of sex hormones including LH,FSH,E2,P,etc.Record the number of eggs,fertilization,embryo quality and so on.?2?Statistical analysis: Quantitative data were analyzed by Shapiro-Wilk method.The quantitative data with the normal distribution were expressed as mean ± standard deviation???? ± s?.The comparison between groups was performed by t test with two independent sample means.One-way analysis of variance,multiple comparisons between groups were performed by LSD method;quantitative data not conforming to normal distribution were expressed by median and quartiles [M?P25,P75?],and Mann-Whitney U test was used for comparison between groups.Multiple sets of comparisons were performed using the Kruskal-Wallis H test.Quantitative data were expressed as rates,and chi-square tests were used for comparison between groups.The test level was ?=0.05.Results1.Comparison of demographic data of the study subjects.Except the blood type?P<0.05?there was no significant difference between the VD deficiency group,insufficiency group and the sufficiency group?P>0.05?.2.Comparison of the serum and follicular fluid levels of vitamin D.There was a positive correlation between the serum and follicular fluid levels of vitamin D,and the correlation coefficient was r=0.832?P<0.01?.3.Comparison of oocyte rate among different groups.There was no significant difference among the three groups or between the two groups in vitamin D deficiency group,insufficiency group and sufficiency group?P>0.05?.4.Comparison of normal fertilization rate,high-qualified embryo rate.?1?Compared with the vitamin D deficiency group and the sufficiency group,there was no statistical difference between two groups?P>0.05?,while high-quality embryo rate was significantly lower in the vitamin D deficiency group than that in sufficiency group?P<0.05?.?2?The normal fertilization rate and high-quality embryo rate were significantly lower in the VD-deficiency group than that in the VD-insufficiency group?P<0.05?.?3?There was no significant difference in normal fertilization rate and high-quality embryo rate between VD-deficiency group and sufficiency group?P>0.05?.Brief Summary Serum levels of 25?OH?D could reflect follicular fluid levels of 25?OH?D,the levels of vitamin D doesn't affect oocyte rate,but affects fertilization rate and high-quality embryo rate.Part ? The Relationship between the levels of vitamin D and gene expression of vitamin D metabolic pathwayObjectives To test the levels of vitamin D and its metabolic genes expression,including CYP2R1,CYP27B1,CYP24A1,GC and vitamin D receptor?VDR?,and explore the relationship between levels of vitamin D/PCOS/insulin resistance and its metabolic pathway genes m RNA expression.Materials and Methods Subjects?1?196 patients were enrolled,who accepted IVF in the Department of Reproductive Medicine at the third Affiliated Hospital of Zhengzhou University from December 2018 to January 2019.69 patients were selected according to the levels of follicular fluid vitamin D in serum,to test the expression of its metabolic pathway gene m RNA in granular cells.?2?Groups:?i?according to the levels of vitamin D in follicular fluid,patients were divided into: VD deficiency group?25?OH?D?50nmol/L,32 cases?,insufficiency group?50nmol/L < 25?OH?D < 75nmol/L,23 cases?and sufficiency group?25?OH?D?75nmol/L,14 cases?.?ii?According to the diagnostic criteria of PCOS,the patients were divided into: PCOS group and non-PCOS group.According to the diagnostic criteria of IR,patients with PCOS were further divided into:PCOS+IR group?23 cases?and PCOS+non-IR group?13 cases?;patients in non-PCOS group were further divided into normal + IR group?11 cases?and normal+ non-IR group?22 cases?.Normal + non-IR group was set as control group.Methods?1?The serum levels of 25?OH?D on the trigger day were determined.The m RNA expression levels of vitamin D metabolic pathway genes CYP2R1,CYP27B1,CYP24A1,GC and VDR in granular cells were determined.?2?Statistical methods: All data were expressed as ???±s,QPCRR original CT values were calculated using the 2-??CT method to calculate the relative m RNA relative expression.The results of different groups were compared using one-way variance.One-way ANOVA was used for analysis.The difference between groups was compared by LSD-T test.The test level was ?=0.05.Results1.Comparison of demographic data of the study subjects.There was no significant difference in age,BMI,infertility age and AMH among the three groups?P>0.05?.2.The relationship between the levels of vitamin D in serum and its metabolic pathway related gene expression.?1?The m RNA expression levels of CYP2R1 and VDR genes were lower in VD deficiency group than in sufficiency group?P<0.05?.There was no significant difference in CYP27B1,CYP24A1 and GC between the two groups?P>0.05?.?2?There was no significant difference in VD metabolic pathway related genes expression between VD-deficiency group and sufficiency group?P>0.05?.3.The relationship between PCOS and IR and gene expression of vitamin D metabolic pathway?1?The comparation of gene expression of vitamin D metabolic pathway in PCOS+non-IR group and control group.There was no significant difference in m RNA expression of CYP2R1,CYP27B1,CYP24A1,GC and VDR between the two groups?P>0.05?.?2?The comparation of gene expression of vitamin D metabolic pathway in PCOS+non-IR group and PCOS+IR group.The CYP2R1 was higher in PCOS+non-IR group than in PCOS+IR group,and the difference was statistically significant?P<0.05?.The gene expression of VDR was higher in PCOS+non-IR group than in PCOS+IR group,but there was no statistically significance?P=0.058?.?3?The comparation of gene expression of vitamin D metabolism pathway in PCOS+IR group and control group.The gene expression levels of CYP2R1 and VDR were lower in PCOS+IR group than in control group?P<0.05?.There was no significant difference in m RNA expression of CYP27B1,CYP24A1,and GC between the two groups?P>0.05?.?4?The comparation of gene expression of vitamin D metabolism pathway in non-PCOS+IR group and control group.The expression of CYP2R1 was significantly lower in non-PCOS+IR group than in control group?P<0.05?.There was no significant difference in m RNA expression of CYP27B1,CYP24A1,GC and VDR between the two groups?P>0.05?.Brief Summary The vitamin D level is closely related to the expression of CYP2R1 gene,and the enzyme that CYP2R1 gene m RNA express is the rate-limiting enzyme of vitamin D.CYP24A1 and GC are the downstream gene of vitamin D metabolic pathway,and the increasing level of vitamin D can't change its m RNA expression.Insulin resistance is a key factor affecting vitamin D metabolic gene expression. |
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