| Controlled ovarian hyperstimulation(COH)plays a crucial role in assisted reproductive technology(ART).In one ovulation induction cycle,multiple eggs can be obtained to form multiple embryos,which increases the chance of embryo transfer and improves the success rate of ART.However,COH cycles are associated with a higher risk of low birth weight(LBW)compared to natural cycles.The risk of low birth weight was significantly higher after fresh embryo transfer than after frozen embryo transfer.This suggests that exposure of the endometrium to a nonphysiological hormone environment after COH may be an important factor leading to low birth weight in fresh cycle embryo transplantation.However,the exact mechanism by which COH increases the risk of low birth weight by affecting the endometrial microenvironment remains unclear.After embryo implantation,endometrial decidual cells,immune cells and foetal chorionic trophoblastic cells form a maternal-foetal interface to adapt to pregnancy.The most abundant immune cells in the maternal interface are uterine natural killer(u NK)cells,which play an important role in embryo implantation and foetal development.However,in the COH cycle,the endometrium is exposed to a nonphysiological hormone environment,and whether the recruitment and function of u NK cells are changed in this environment,affecting placental angiogenesis and the growth and development of the foetus,is still unclear and needs further investigation.In this study,to exclude the influence of COH on oocytes and early embryos,wedesigned a surrogate mouse embryo transfer model using different levels of ovulatory drugs.We focused on the effect of COH on u NK cells and the related mechanism of low foetal weight in mice.The following research results were obtained:Part 1:The effect of controlled ovarian hyperstimulation on fetal and placental development at 14.5 days gestation in miceObjective:To exclude the influence of COH on oocytes and early embryos,we established a mouse embryo transfer model to explore the influence of COH on foetal mice at 14.5 days of gestation by affecting the uterine microenvironment.Methods:Pseudopregnant mice were divided into three groups,natural conception(NC),low-dose controlled stimulation(L-COH)and high-dose controlled hyperstimulation(H-COH).To exclude the influence of COH on oocytes and early embryos,3.5-day blastocysts from natural gestation were transplanted into three groups of surrogate mothers 3.5 days after mating between surrogate mothers and ligated male mice.In this way,we elucidated the influence of COH on foetal development by influencing intrauterine environment.Mouse foetuses were collected at 14.5 days of gestation from surrogate mothers.We analysed pregnancy outcomes in the three groups of surrogate mothers and compared the embryo implantation rate,foetal survival rate,foetal formation rate,and foetal weight.According to the above data,we analysed the influence of COH on foetal and placental development.Results:(1)The embryo implantation rate refers to the ratio of the number of implanted embryos to the total number of transplanted embryos.The implantation rates of the NC,L-COH and H-COH groups were 87.5±12.9(%),68.8±14.3(%)and 41.4±12.5(%),respectively.The implantation rates of the L-COH and H-COH groups were significantly lower than the NC group(P<0.01),and the implantation rate of the H-COH group was significantly lower than the L-COH group(P<0.05).(2)Foetal survival rate refers to the ratio of the number of live births to the total number of transplanted embryos.The survival rates of the NC,L-COH and H-COH groups were81.3±12.0(%),62.5±15.9(%)and 32.0±9.6(%),respectively.The survival rates of the L-COH and H-COH groups were significantly lower than the NC group(P<0.01),and the survival rate of the H-COH group was significantly lower than the L-COH group(P<0.05).(3)The foetal formation rate refers to the ratio of the number of live births to the number of implanted embryos.The foetal formation rate was compared,and there was no significant difference among the three groups(P>0.05).(4)The weight of the foetus was analysed,demonstrating that the foetal weights of the NC,L-COH and H-COH groups were 308±32 mg,226±55 mg and 180±54 mg,respectively.The foetal weights of the L-COH and H-COH groups were significantly reduced compared to the NC group(P<0.01),and the foetal weight of the H-COH group was significantly reduced compared to the L-COH group(P<0.05).Conclusions:COH may affect the developmental potential of embryos by affecting the endometrial microenvironment,leading to a significant decrease in the embryo implantation rate and foetal survival.Moreover,the higher the dose of ovulatory hormone,the lower are the embryo implantation and foetal survival rates.COH might lead to foetal intrauterine growth restriction by affecting the endometrial microenvironment.Furthermore,hormone administration and foetal weight were inversely correlated,with higher doses of ovulatory hormone yielding lower foetal weights.Part 2:The effect of controlled ovarian hyperstimulation on the structure and function of placental tissues at 14.5 days of gestation in miceObjective:In a surrogate mouse embryo transfer model using different levels of ovulatory drugs,we investigated the effect of COH on the structure and function of placental tissues at 14.5 days of pregnancy in mice by influencing the uterine microenvironment.Methods:In the same part,surrogate mice were divided into three groups:NC,L-COH and H-COH.The placentas of mice were collected on 14.5 days of gestation from surrogate mothers,and placental weights were analysed.The effects of COH on the structure of the placenta were analysed by haematoxylin-eosin staining and periodic acid-Schiff staining.CD31 and laminin expression was detected by tissue immunohistochemistry,and the effect of COH on placental vascular density was analysed.RT-q PCR was used to detect relative expression levels of 6 paternally imprinted genes and12 maternally imprinted genes and the relative expression levels of 14 placental nutrient transport channel genes in three groups of mouse placentas to clarify the influence of COH on placental structure and function.Results:(1)The weights of placentas were analysed,revealing that placental weights of the NC,L-COH and H-COH groups were 107±15 mg,110±17 mg and 101±18 mg,respectively.There were no significant differences among the three groups(P>0.05).(2)Analysis of placental efficiency:The placental efficiencies in the NC,L-COH and H-COH groups were 2.833±0.278,2.056±0.376 and 1.828±0.471,respectively.Compared to the NC group,placental efficiencies in the L-COH and H-COH groups were significantly reduced(P<0.01).Compared to the L-COH group,the placental efficiency of the H-COH group also showed a downward trend,but there was no significant difference between the two groups(P>0.05).(3)Analysis of the PAS staining area of placentas:The ratio of the PAS staining-positive area to the whole placental area in the NC,L-COH and H-COH groups was 14.364±5.381,20.182±3.010 and 26.455±4.560,respectively.Compared to the NC group,the ratio of the PAS-positive area to the whole placental area in the L-COH and H-COH groups was significantly higher(P<0.05,P<0.01).Compared to the L-COH group,the area of the PAS staining-positive area in the H-COH group was increased,but this difference was not significant(P>0.05).(4)Immunohistochemical analysis of placental CD31:The MVDs of placental microvessels in the NC,L-COH and H-COH groups were 510.3±58.0,443.2±72.8 and400.3±74.9,respectively.Compared to the NC group,MVDs in the L-COH and H-COH groups were significantly reduced(P<0.05 and P<0.01).Moreover,MVDs in the H-COH group were reduced compared to the L-COH group,but there was no significant difference between the two groups(P>0.05).(5)Immunohistochemical analysis of placental laminin:MVDs of placental microvessels in the NC,L-COH and H-COH groups were 329.3±64.8,276.5±39.4 and254.9±60.3,respectively.Compared to the NC group,MVDs in the L-COH and H-COH groups were significantly reduced(P<0.05 and P<0.01).Moreover,MVDs in the H-COH group were lower than in the L-COH group,but there was no significant difference between the two groups(P>0.05).(6)The expression of imprinted genes in mouse placental tissue:Parentally imprinted gene expression:Relative expression levels of Igf2,Kcnq1ot1 and Snrpn in the H-COH group were significantly downregulated compared to those in the NC and L-COH groups(P<0.05).Compared to NC,relative expression levels of Kcnq1ot1 in the L-COH group were also significantly downregulated(P<0.05).Expression of maternally imprinted genes in placental tissues:Compared to the NC group,expression of 4 genes(Gatm,Dcn,Osbpl5,and Phlda2)in the L-COH and H-COH groups was significantly upregulated.Compared to the L-COH group,H-COH expression was also significantly upregulated,and the above differences were statistically significant(P<0.05).Compared to the NC and L-COH groups,the expression of four genes(Ascl2,Peg3,Cd81,and Ube3a)in the H-COH group was significantly upregulated(P<0.05).(7)Expression of nutrition channel genes in placentas:There were no significant changes in the twelve examined nutrition channel genes(Slc26a7,Slc2a1,Slc2a3,Slc38a1,Slc38a2,Slc3a2,Slc6a6,Slc19a2,Slc22a18,Slc22a3,Slc40a1,and ATP1a1)among the three groups(P>0.05).Conclusions:COH significantly reduced placental efficiency,and COH resulted in a significant increase in the PAS staining-positive region in mouse placentas.Some sponge trophoblast cells invaded the trophoblast layer of the vestibular canal,resulting in abnormal morphology of placental tissues.COH may be an important cause of LBW,as it reduces the density of microvessels in the labyrinthus layer of mouse placentas.Part 3:Study on the mechanism of COH-induced low birth weight in mice by changing the recruitment of decidual u NK cellsObjective:To explore the mechanism of COH’s influence on the recruitment of decidual u NK cells in mice.Methods:The experimental design is the same as in Part 1.Decidual tissues of the three groups were collected at 8.5 days of pregnancy,and the size,shape and weight of decidual tissues were analysed.DBA~+u NK cells in decidual tissues of the three groups were identified by DBA fluorescent staining,and DBA~+u NK cell densities were compared among the three groups.Fluorescence quantitative polymerase chain reaction(RT-QPCR)was used to detect m RNA expression levels of angiogenic cytokines,promoting foetal growth,tissue remodelling cytokines and immune cytokines in the three groups.The protein expression levels of these cytokines secreted and regulated by u NK cells were measured by magnetic Luminex performance multifactor chip detection technology.These were compared among the three groups.Results:(1)Morphological analysis of the decidua in mice at 8.5 days of gestation showed that the morphology of the decidua in the NC group was full and uniform.The morphology of the decidua in the L-COH group was slightly smaller,and the size was not equal.However,morphology in the H-COH group was significantly smaller and varied in size.Decidual weight analysis:The decidual weight at 8.5 days of gestation in the NC,L-COH and H-COH groups was 28.3±4.95 mg,25.3±7.66 mg,and 19.2±7.57 mg,respectively.The decidual weight of the L-COH and H-COH groups was significantly lower than in the NC group(P<0.001),and the weight of the H-COH group was significantly lower than the L-COH group(P<0.01).(2)The analysis of cell densities of decidual u NK revealed the following:DBA~+u NK cell densities(cell number/total cell number under 20x80 amplification)were 27.9±7.2(%),13.7±4.4(%)and 10.9±4.3(%)in the NC,L-COH and H-COH groups,respectively.Densities of DBA~+u NK cells in the L-COH and H-COH groups were significantly lower than in the NC group(P<0.001).The density of DBA~+u NK cells in the H-COH group was lower than in the L-COH group,but the difference was not statistically significant(P>0.05).(3)m RNA expression levels of secreted angiogenic factors and related cytokines by u NK cells:Compared to the NC group,the expression levels of Pgf,Vegfb,Vegfd and Hifla were significantly decreased in the L-COH and H-COH groups(P<0.05),and the m RNA expression levels of these factors were also significantly decreased in the H-COH and the L-COH groups(P<0.05).Compared to the NC group,the expression levels of Vegfa,Kdr,Flt4,Angpt2 and Angpt4 in the decidual tissue of the H-COH group were significantly downregulated(P<0.05),while there was no significant change in the L-COH group(P>0.05).(4)m RNA expression levels of secreted factors promoting foetal growth and tissue remodelling and related cytokines by u NK cells:The expression levels of Opn,Ogn,Csf2,Lif,Tgf-β1,Igfbp1,Tek,IL-1A and Nfil3 in the L-COH and the H-COH groups were all downregulated compared to the NC group,and the expression levels of Opn,Ogn,Csf2,Lif,Tgf-β1 and Igfbp1 in the H-COH group were also significantly downregulated compared to the L-COH group.Compared to the NC group,the expression level of CSF-1was significantly downregulated in the H-COH group.Compared to the NC and L-COH groups,the expression level of Cxcl12 was significantly downregulated in the H-COH group.There was no significant difference in the expression level of Ptn among the groups.However,compared to the NC group,the expression level of Cxcl10 was upregulated in both the L-COH and the H-COH groups and was significantly upregulated in the H-COH group compared to the L-COH group.The above differences were all statistically significant(P<0.05).(5)m RNA expression levels of secreted immune factors and related cytokines by u NKcells:Compared to the NC group,the expression levels of Cd96,Ltf,Eomes and Stap1were significantly decreased in the L-COH and H-COH groups(P<0.05).Compared to the NC group,the expression levels of Rac2 and Batf were significantly decreased in the L-COH and H-COH groups(P<0.05).Compared to the NC and L-COH groups,the expression level of Rab27a was significantly decreased in the H-COH group(P<0.05).(6)Protein expression levels of secreted and related cytokines by u NK cells:Compared to the NC group,protein expression levels of Csf3 and Il-12 P70 were downregulated in both the L-COH and H-COH groups and were significantly downregulated in the H-COH group compared to the L-COH group(P<0.05).Compared to the NC group,protein expression levels of Il-1a,Il-13,Ccl4,Il-4,Opn,Kdr and IFN-γwere significantly downregulated in the L-COH and H-COH groups(P<0.05).Compared to the NC group,protein expression levels of Il-1b,Il-10,Ccl5,Il-6,Cxcl12 and Tnf were significantly downregulated in only the H-COH group(P<0.05).The protein expression levels of Ccl3,Cxcl16 and M-CSF showed no significant differences among the groups(P>0.05).Conclusions:COH may reduce decidual weight by affecting the intrauterine environment,and the higher the hormone dose is,the lower the weight.COH may significantly reduce the densities of DBA~+u NK cells in decidual tissues,and the higher the hormone dose is,the fewer the DBA~+u NK cells.m RNA and protein expression of angiogenic cytokines,growth promoting foetal and tissue remodelling cytokines and immune cytokines secreted and released by u NK cells were significantly downregulated.COH may reduce the density of decidual u NK cells by affecting the recruitment of decidual u NK cells and may reduce the levels of cytokines produced by u NK cells,affecting the development and function of the placenta and resulting in low birth weight in mice. |
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