CN106018825A - Method for improving test-tube baby clinical outcome by selecting embryo with good development potential - Google Patents
Method for improving test-tube baby clinical outcome by selecting embryo with good development potential Download PDFInfo
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Abstract
The invention discloses a method for improving the test-tube baby clinical outcome by selecting an embryo with good development potential. The method comprises five steps that the transplanting embryo with good development potential is selected mainly according to IL-8 in an embryo culture solution before the transplanting; concretely, a liquid phase chip high-flux protein analysis technology is used for detecting the content of IL-8 in a sample; an IL-8 positive embryo in the culture solution is selected to be combined with a morphology evaluation method to perform transplantation; the IL-8 positive embryo in the culture solution before the transplantation is used for being combined with the morphological high-quality embryo to perform transplantation; the pregnancy rate can be improved by more than 8 percent, and can reach 60.76 percent; compared with the existing transplantation method for selecting the embryo singly by a morphology method, the method provided by the invention has the advantages that the pregnancy rate can be improved by more than 8 percent to 12 percent. When the method is combined with the existing morphology method for selecting high-quality embryo; or the high-quality embryo and the ordinary embryo are transplanted to an in vitro fertilization person, the embryo planting rate can also be improved; the baby live birth number is increased; the patient clinic pregnancy rate is improved; the abortion rate is reduced.
Description
Technical field
The present invention is belonging to reproductive medicine science, test-tube baby's fields of implantation, is specifically related to carrying out test-tube baby's transplanting
Before, select by cultivating the content of interleukin 8 (IL-8) in the culture fluid treating transplanting embryo before measuring Human embryo transplanting
The transplanting embryo that potentiality of development is good, to improve pregnancy outcome.
Background technology
Along with auxiliary procreation technology, alternatively tube-test baby techniques increases in worldwide application, the fastest but also non-wound
Best transplanting embryo is selected to wound property to remain the major issue that reproductive medicine faces.Great majority are carried out test-tube baby
Patient for, for obtaining higher Embryo Implantation Rate in (IRs) and pregnancy rate, transplant every time and need to transplant multiple embryo, and transplant
Multiple embryos can cause multiple pregnancy.The increase of the multiple pregnancy caused by external fertilization (IVF/IVSI), makes society face one to be
Arrange great health problem, and increase the weight of the burden of hygiene department.Medical science multiple pregnancy brought along with people and society
The understanding of economic problems increases, and prevention multiple pregnancy has become a central task of auxiliary procreation technology.Selectivity list embryo
Transplanting is considered as to prevent the unique selection of multiple pregnancy in IVF cycles.Not reducing of single tire transplanting should be selected pregnant
Be pregnent rate, be necessary for improving embryo IRs and find a kind of identify transplanting before the method for embryo of the highest plantation potential.At present, form
Learn Evaluation Method and remain the main method evaluating embryo quality.Morphological assessment method is mainly according to cell number and the shape thereof of embryo
State outward appearance selects transplanting embryo, and this method can not predict the plantation potential of embryo well.Owing to commenting with morphology
Estimate method evaluation evaluation embryo quality has certain limitation, many researcheres seeking other of evaluation embryonic germ potential always
Method.Glucose that the method (embryo transfer) evaluating embryo currently studied includes measuring in embryo medium, lactic acid,
Acetone acid or amino acid whose content；Measure the oxygen consumption of embryo；And the method such as genomics and protein science.In recent years, non-
Invasive methods is considered as Clinical Outcome's method the most likely after prediction auxiliary procreation technology.The existing non-wound of researcher
The several metabolism indexs of wound property method detection embryonic development, and testing result shows, metabolite and patient in embryo medium
Clinical Outcome be correlated with.Being imaged by time difference microscope (time-lapse device), embryo's observed in atraumatic ground
Grow, be a kind of better method evaluating embryo quality；Existing research shows, selects embryo to move in this way
Plant, it is possible to obtain good Clinical Outcome.But, owing to lacking large sample and random clinical research, researcher is difficult to judge
Select embryo's method can improve Clinical Outcome with kinetic parameter, and be difficult to judge that any kinetic parameter is evaluating embryo's matter
In amount more valuable.
Cytokine is an albuminoid of emiocytosis.Under physiology or pathological condition, this albumen as hormone regulating and controlling device,
Can regulating cell and the function of tissue under nanomole to picomole (nano to picomolar) concentration.Cytokine
Basic function is to change a large amount of cells and the performance of tissue.Cytokine can be passed through autocrine, paracrine or secrete at a distance
(under particular case) works.In cytokine and cell membrane, specific receptor combines, the cascade reaction of inducement signal path, knot
Fruit causes cell and tissue generating effect.At reproductive immunology, cytokine is not only involved in menstruation, ovulates, gives a birth and Embryo implantation
Deng physiological process, also participate in the pathological process such as premature labor and endometriosis.
For many years, researcher attempt always with the cytokine in embryo medium before transplanting predict embryo quality and
Pregnancy outcome.The nineties in 20th century, scholar detects interleukin-1 in Human embryo culture fluid (I L-1), interleukin-6
(IL-6), colony-stimulating factor-1 (CSF-1), the containing of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β)
Amount, and propose to predict the probability of patient IVF/IVSI pregnancy outcome by these cytokines in embryo medium.?
2012, a document showed, gravid woman's group compares with unrecognizing factor, neoplasm necrosis in embryo medium before human transplant
The concentration of factor-alpha (TNF-α) is organized relatively low gravid woman, and leukaemia inhibitory factor in embryo medium before human transplant
(LIF) the highest in gravid woman's group, disclose LIF and be probably the factor required for Embryo implantation, and TNF-α can be as prediction embryo
An index unsuccessfully planted by tire.And we detect the mankind with liquid-phase chip high-throughout protein analysis technology (LHTPA)
Transplanting TNF-α, the content of IL-6, IL-1 β and GM-CSF in front embryo medium, result does not find this several cytokines
Can predict potentiality of development and the Clinical Outcome of embryo, therefore this data is not delivered.This researcher continues research and measures people's embryo
The content of interleukin 8 (IL-8) in the culture fluid treating transplanting embryo cultivated by tire before transplanting, and analyze and study the dense of IL-8
Degree and embryo quality and the relation of pregnancy outcome, obtained encouraging achievement.
Summary of the invention
It is an object of the present invention to provide the new method of a kind of transplanting embryo quality evaluating external fertilization receptor.The present invention is led to
Cross research to draw, measure to cultivate before Human embryo is transplanted and treat that in the culture fluid of transplanting embryo, the content of IL-8 is to select potentiality of development good
The method of good embryo, to improve the Clinical Outcome of external fertilization receptor.
This method comprises the following steps:
(1) on the same day transplanted, collect the cultivation before each embryo transfer of each external fertilization receptor and treat transplanting embryo
Culture fluid.First day of after fertilization in vitro, is confirming after fertilization, by each germ cell of each external fertilization receptor respectively
It is placed in the spilting of an egg liquid drop of 30-50 μ L 10% human serum albumin, puts 37 DEG C, 5%CO2Incubator in continue cultivation 2
My god, to embryo transfer；Now, embryo cultivated the most to the 3rd day, and the 4h-6h before carrying out embryo transfer, according to ovum
The morphological assessment method based on rate and morphology of splitting is evaluated each external fertilization receptor and is treated the quality of transplanting embryo, each external
Fertilization receptor transplants 2-3 embryo every time；Each external fertilization receptor after the embryo transplanted is selected, by embryo to be transplanted
Remove, i.e. collect to cultivate and treat the transplanting embryo spilting of an egg liquid drop containing 10% human serum albumin of 2 days, i.e. cultivate and treat Transplanted Embryonic
The culture fluid of tire；In view of each external fertilization receptor transplants 2-3 embryo every time, each embryo is both placed in one before transplantation
30-50 μ L cultivates 2 in the spilting of an egg liquid drop containing 10% human serum albumin, each embryo transfer of each external fertilization receptor
Before cultivation treat the culture fluid of transplanting embryo be cultivate respectively 2-3 treat transplanting embryo 2 days containing 10% human serum albumin's
Spilting of an egg liquid drop, cultivates this 2-3 the drop treating transplanting embryo, composition before collecting each each embryo transfer of external fertilization receptor
A specimen, every a specimen is containing 25 μ L-40 μ L culture fluid；It is collected under similarity condition the cultivation not placing embryo cultivated
Liquid is as negative control；
(2) interleukin 8 in every a specimen is detected with liquid-phase chip high-throughout protein analysis technology (LHTPA)
The content of element-8 (IL-8)；
(3) transplanting embryo is selected according to the content of interleukin 8 (IL-8) in morphological assessment method and specimen, will
The transplanting embryo selected is transplanted to external fertilization receptor；Available transplanting embryo morphology is improved quality same or similar, then select it
In culture fluid, IL-8 is positive, and i.e. in culture fluid, IL-8 concentration range embryo between 0.19pg/ml to 10.32pg/ml enters
Row is transplanted；
(4) if available transplanting embryo is improved quality to morphology differ or mutually the most similar, then select IL-in its culture fluid
8 is positive, i.e. IL-8 concentration range embryo between 0.19pg/ml to 10.32pg/ml and/or select the form of the foetus state in culture fluid
The preferable embryo that improves quality transplants.
(5) time detecting IL-8 from making a collection of specimens to is 4h-6h, therefore i.e. can use the method on the same day of embryo transfer
Screen the preferable embryo of potentiality of development to transplant.
The invention has the beneficial effects as follows, select potentiality of development good according to the cytokine in embryo medium before transplanting
Transplanting embryo, to improve pregnancy outcome.Gravid woman's group compares with unrecognizing factor, swollen in embryo medium before human transplant
The concentration of tumor necrosis factor α (TNF-α) is organized relatively low gravid woman, and before human transplant in embryo medium leukemia suppression because of
Son (LIF) is the highest in gravid woman's group, and disclosing LIF is the factor required for Embryo implantation, and tumor necrosis factor α (TNF-
α) can be as an index of prediction Embryo implantation failure.Excellent with transplanting in morphology positive for IL-8 in forefathers' embryo medium
Matter embryo transfer can improve more than 8% to IVF/ICSI patient, pregnancy rate (pregnancy rate), reach 60.76%；With transplanting
Embryo quality positive for IL-8 in forefathers' embryo medium and common embryo transfer can improve to IVF/ICSI patient, pregnancy rate
More than 12%, reach 47.37%.Thus, combining existing morphological method by this method selects embryo to transplant, more existing
Alone morphological method select embryo transplant, pregnancy rate can improve more than 8-12%.Combine existing by this method
Morphological method selects embryo quality, or embryo quality and common embryo transfer are to IVF/ICSI patient, it is also possible to improve embryo
Implantation Rate, increases baby's survival rates of patient, improves patient clinical pregnancy rate and reduces the abortion ratio of patient.
Accompanying drawing explanation
Fig. 1. the concentration (IL-8concentration) of IL-8 in the culture fluid transplanting front embryo is measured by LHTPA method.
* P=0.003,0.000 and 0.000, with negative control group (negative control group), seminar
(study group) and LI-8 feminine gender group (LI-8negative group) compare.P=0.000, compares with LI-8 feminine gender group
(LI-8negative group).Seminar is divided into LI-8 positive group by the height of the value according to the LI-8 detected in sample
(LI-8positive group) and LI-8 feminine gender group, in LI-8 positive group, the equal height of the value of the LI-8 detected in sample
In 0.19pg/ml, and in LI-8 feminine gender group, the value of the LI-8 detected in sample be below 0.19pg/ml.Every a mark
This is made up of the culture fluid of all transplanting embryos in this fresh transplanting cycle of each patient；It is collected under similarity condition cultivation
Do not place the culture fluid of embryo as negative control.Result represents with mean ± standard error (mean ± SEM).The quilt that n=is total
Detection sample number.
Fig. 2. the Clinical Outcome of external fertilization receptor is predicted with the IL-8 in the culture fluid transplanting front Human embryo
Gestation between LI-8 positive group (LI-8positive group) and LI-8 feminine gender group (IL-8negative group)
Rate (pregnancy rate), Embryo Implantation Rate in (implantation rate), Clinical Pregnancy Rate in (clinical
Pregnancy rate), abortion ratio (abortion rate), biochemical pregnancies rate (chemical pregnancy rate) and
Live birth rate (live-birth rate) compares.
P=0.022, * P=0.020, compares with LI-8 feminine gender group.
Detailed description of the invention
Implementation, this method comprises the following steps:
(1) on the same day transplanted, collect the cultivation before each embryo transfer of each external fertilization receptor and treat transplanting embryo
Culture fluid.First day of after fertilization in vitro, is confirming after fertilization, by each germ cell of each external fertilization receptor respectively
It is placed on 30-50 μ L 10% human serum albumin (SAGE In-Vitro Fertilization, Inc.；Trumbull,CT,
USA) spilting of an egg liquid (ART-1026, SAGE In-Vitro Fertilization, Inc.；Trumbull, CT, USA) drop
In, put 37 DEG C, 5%CO2Incubator in continue cultivate 2 days, to embryo transfer；Now, embryo has cultivated the most to the 3rd
My god, the 4h-6h before carrying out embryo transfer, evaluates every individual according to the morphological assessment method based on cleavage rates and morphology
Outer fertilization receptor treats the quality of transplanting embryo, and each external fertilization receptor transplants 2-3 embryo every time；Each external fertilization receptor
After the embryo transplanted is selected, embryo to be transplanted is removed, i.e. collect cultivate treat transplanting embryo 2 days containing 10% human blood
Pure albumen (SAGE In-Vitro Fertilization, Inc.；Trumbull, CT, USA) spilting of an egg liquid (ART-1026,
SAGE In-Vitro Fertilization,Inc.；Trumbull, CT, USA) drop (cultivation of transplanting embryo is treated in cultivation
Liquid).In view of each external fertilization receptor transplants 2-3 embryo every time, each embryo is both placed in a 30-50 μ L before transplantation
Containing 10% human serum albumin (SAGE In-Vitro Fertilization, Inc.；Trumbull, CT, USA) spilting of an egg liquid
(ART-1026,SAGE In-Vitro Fertilization,Inc.；Trumbull, CT, USA) drop is cultivated 2, each
Cultivation before each embryo transfer of external fertilization receptor treats that the culture fluid of transplanting embryo treats transplanting embryo for cultivating 2-3 respectively
2 days containing 10% human serum albumin (SAGE In-Vitro Fertilization, Inc.；Trumbull, CT, USA) ovum
Split liquid (ART-1026, SAGE In-Vitro Fertilization, Inc.；Trumbull, CT, USA) drop, collect each
This 2-3 the drop treating transplanting embryo is cultivated before each embryo transfer of external fertilization receptor, a specimen of composition, every a specimen
Containing 25 μ L-40 μ L culture fluid；It is collected under similarity condition the culture fluid not placing embryo cultivated as negative control.
External fertilization receptor: in January, 2014 to December, carries out the Infertile couples of external fertilization treatment in my center.Carry out
The indication of assisted reproductive therapy includes that Tubal factor, endometriosis, male factor or idiopathic are infertile and exempt from
Epidemiology factor or front test-tube baby transplant cycle unknown cause loser.
(2) interleukin 8 in every a specimen is detected with liquid-phase chip high-throughout protein analysis technology (LHTPA)
The content of element-8 (IL-8).
(3) transplanting embryo is selected according to the content of interleukin 8 (IL-8) in morphological assessment method and specimen, will
The transplanting embryo selected is transplanted to external fertilization receptor；Available transplanting embryo morphology is improved quality same or similar, then select it
In culture fluid, IL-8 is positive, and i.e. in culture fluid, IL-8 concentration range embryo between 0.19pg/ml to 10.32pg/ml enters
Row is transplanted.
(4) if available transplanting embryo morphology is improved quality differ or mutually the most similar, then select IL-8 in its culture fluid
IL-8 concentration range embryo between 0.19pg/ml to 10.32pg/ml and/or select tire morphology in the positive, i.e. culture fluid
The preferable embryo that improves quality transplants.
(5) time detecting IL-8 from making a collection of specimens to is 4h-6h, therefore i.e. can use the method on the same day of embryo transfer
Screen the preferable embryo of potentiality of development to transplant.
The present invention is further illustrated below in conjunction with specific embodiment,
Embodiment 1
2714 (medical record number) Infertile couples wife's side name, old X, 33 years old, bridegroom's or husband's side name, Qin XX, diagnosis: infertility:
It is postoperative that left fallopian tube blocks Lap；After intrauterine adhesion hysteroscope operation；The wife's side took ovum on January 17th, 2014, January 20 in 2014
Day row embryo transfer operation；In the culture fluid of embryo transfer operation consent of being expert at mensuration transplanting embryo, the concentration of IL8 is 0.21pg/
Ml, institute's transplanting embryo rank is 2/5 and 2/7；It is positive, in JIUYUE 25 in 2014 that wife's side row embryo transfer Post operation surveys blood hCG in 14 days
Day Cesarean esction one work boy baby, body weight 3.2 kilograms.
Embodiment 2
5124 (medical record number) Infertile couples wife's side name, poplar XX, 30 years old, bridegroom's or husband's side name, side X, diagnosis: fertility:
After pelvic cavity severe adhesion exclusion；Pelvic cavity chamber tuberculosis (old)；The wife's side took ovum on January 18th, 2014, January 21 in 2014
Day row embryo transfer operation；In the culture fluid of embryo transfer operation consent of being expert at mensuration transplanting embryo, the concentration of IL8 is 0.27pg/
Ml, institute's transplanting embryo rank is 1/5 and 1/7；It is positive, October 5 in 2014 that wife's side row embryo transfer Post operation surveys blood hCG in 14 days
Day Cesarean esction one work boy baby, body weight 3.1 kilograms.
Embodiment 3
5120 (medical record number) Infertile couples wife's side name, willow X, 27 years old, bridegroom's or husband's side name, thank to X, diagnosis: right side fallopian tube
After gestation expectant treatment；Left side tubal pregnancy is guarded postoperative；RSA；The weak asthenoteratozoospermia of husband；The wife's side was on January 19th, 2014
Take ovum, in row embryo transfer operation on January 22nd, 2014；Embryo transfer operation consent of being expert at measures in the culture fluid of transplanting embryo
The concentration of IL8 is 0.55pg/ml, and institute's transplanting embryo rank is 1/8 and 1/7；Wife's side row embryo transfer Post operation surveys blood hCG in 14 days
The positive, in Cesarean esction one boy baby alive on October 4th, 2014, body weight 3.5 kilograms.
Embodiment 4
5110 (medical record number) Infertile couples wife's side name, congratulates XX, bridegroom's or husband's side name, dragon XX, diagnosis: fertility: right side
Obstruction of fallopian tube Lap is postoperative；The bilateral ovaries Lap that windows is postoperative；The wife's side took ovum on January 19th, 2014, on January 22nd, 2014
Row embryo transfer is performed the operation；In the culture fluid of embryo transfer operation consent of being expert at mensuration transplanting embryo, the concentration of IL-8 is 0.52pg/ml,
Institute's transplanting embryo rank is 1/8 and 1/8；It is positive, on October 4th, 2014 that wife's side row embryo transfer Post operation surveys blood hCG in 14 days
Cesarean esction one girl baby alive, body weight 3.15 kilograms.
Embodiment 5
5119 (medical record number) Infertile couples wife's side name, pays XX, bridegroom's or husband's side name, old XX, diagnosis: infertility: ovary
Reserve function goes down；Husband's azoospermia；Hepatitis B virus carriers；The wife's side takes ovum on January 20th, 2014, in January, 2014
Row embryo transfer operation on the 23rd；In the culture fluid of embryo transfer operation consent of being expert at mensuration transplanting embryo, the concentration of IL-8 is
6.58pg/ml, institute's transplanting embryo rank is 1/8 and 1/8；It is positive, in 2014 that wife's side row embryo transfer Post operation surveys blood hCG in 14 days
Cesarean esction one work on October 9, in girl baby, body weight 2.9 kilograms.
Embodiment 6
5769 (medical record number) Infertile couples wife's side name, XX slowly, bridegroom's or husband's side name, fertility: husband's azoospermia；Month
Through uncomfortable；One time AIH is postoperative；The wife's side takes ovum on July 12nd, 2014, in row embryo transfer operation on July 15th, 2014；It is expert at
In the culture fluid of embryo transfer operation consent mensuration transplanting embryo, the concentration of IL-8 is 0.52pg/ml, and institute's transplanting embryo rank is 1/8
With 1/8；It is positive that wife's side row embryo transfer Post operation surveys blood hCG in 14 days, and showed no increases in output a girl baby alive and a man alive on October 9th, 2014
Baby, body weight is respectively 1.99 kilograms and 1.73 kilograms.
Effect is comprehensively described when 330 example:
(1) before transplanting, the culture fluid of Human embryo can detect IL-8.LI-8 in the culture fluid of Human embryo before transplanting
Positive rate is 32.42% (107/330), and the positive rate of negative control group LI-8 is 0% (0/9), (table 1 and Fig. 1).
Table 1. people transplants the concentration of IL-8 in front embryo medium
Note: * P=0.003,0.000 and 0.000, compare with negative control group, seminar and LI-8 feminine gender group.P=
0.000, compare with LI-8 feminine gender group.
(2) IL-8 in embryo medium and the relation of external fertilization receptor's final result before transplanting
It is not 54.21% (58/107) and 27.62% (66/239) at LI-8 positive group, pregnancy rate and Embryo Implantation Rate in；
It is not 40.81% (91/223) and 19.92% (97/487) at LI-8 feminine gender group, pregnancy rate and Embryo Implantation Rate in；LI-8 is positive
Group and between LI-8 feminine gender group the most all there were significant differences the property (P=0.022 and 0.020) of pregnancy rate and Embryo Implantation Rate in；?
LI-8 positive group compares with LI-8 feminine gender group, and Clinical Pregnancy Rate in is higher, and abortion ratio is relatively low, but being not significantly different from property between two groups
(P=0.12 and 0.17)；Between LI-8 positive group with LI-8 feminine gender group, biochemical pregnancies rate compares with live birth rate, being not significantly different from property
((table 2 and Fig. 2).Result shows, transplanting the indication external fertilization receptor patient of the IL-8 positive in forefathers' embryo medium has higher
Pregnancy rate and preferable Embryo implantation potential, also indication external fertilization receptor may have high Clinical Pregnancy Rate in and relatively low stream
Productivity.Transplant external fertilization receptor positive for IL-8 in forefathers' embryo medium, although the patient that its live birth rate is negative with IL-8
Relatively being not significantly different from property, but because its pregnancy rate increases, result to its baby's survival rates increases.If with transplanting front Human embryo
(((mean+2SD) predicts external fertilization receptor after fertilization in vitro to 0.19 to 10.32pg/ml to the concentration of the IL-8 in culture fluid
The most pregnant, then positive predictive value=56.86% (58/ (58+49-5)).
Table 2. compares the clinic of the external fertilization receptor transplanting patient positive with negative for IL-8 in forefathers' embryo medium
Final result
P=0.022, * P=0.020
(3) relation between IL-8 and the quality of transplanting embryo and cell number in forefathers' embryo medium is transplanted
Between LI-8 positive group and LI-8 feminine gender group, the average cell number (mean ± SEM) of transplanting embryo does not has obvious difference
(P=0.32) (table 3).Transplant patient positive with negative for IL-8 in forefathers' embryo medium, be divided into according to transplanting embryo quality
Three groups: embryo quality group (good quality embryo group) high-quality and common embryo's group (good and ordinary
Quality embryo group) and common embryo's group (ordinary quality embryo group).At embryo quality
Group, the embryo of all graft is embryo quality；In high-quality and common embryo's group, existing in the embryo of every graft
Embryo quality, has again common embryo；In common embryo's group, the embryo of every graft is common embryo.Result shows,
The being not significantly different from property of quality (P=0.71) (table 3) of transplanting embryo between LI-8 positive group and LI-8 feminine gender group.From common embryo
Group, high-quality and common embryo's group are to embryo quality group, pregnancy rate (pregnancy between LI-8 positive group and LI-8 feminine gender group
Rate) comparing, diversity is gradually increased, but does not has significance (P=1.00,0.18 and 0.07) (table 4).Result shows, works as shifting
When planting the embryo of equal in quality, transplant the pregnancy rate (pregnancy of the positive relatively IL-8 negative patient of IL-8 in forefathers' embryo medium
Rate) high, when transplanting IL-8 positive transplanting embryo quality (good quality embryo) in forefathers' embryo medium, pregnant
Rate of being pregnent (pregnancy rate) may be up to 60.76% (table 4).
Between table 3.LI-8 positive group and LI-8 feminine gender group, quality and the cell number of transplanting embryo compare
The quality of transplanting embryo and the relation of pregnancy rate between table 4.LI-8 positive group and LI-8 feminine gender group
Transplant to external fertilization receptor with transplanting embryo quality in morphology positive for IL-8 in forefathers' embryo medium, pregnant
Rate of being pregnent (pregnancy rate) can improve more than 8%, reaches 60.76%；With transplanting the IL-8 positive in forefathers' embryo medium
Embryo quality and common embryo transfer can improve more than 12% to external fertilization receptor, pregnancy rate, reach 47.37%.Thus, with this
Method combines existing morphological method and selects embryo to transplant, and more existing alone morphological method selects embryo to enter
Row is transplanted, and pregnancy rate can improve more than 8-12%.
(4) analyze: why can select, by this method, transplanting embryo that potentiality of development is good and improve external fertilization and be subject to
The pregnancy outcome of person
Embryo be implanted as successful pregnancy institute required, about half of embryo miscarries after the implantation.Clinically,
Embryonic limb bud cell failure is closely related with the low success rate of recurrent abortion and external fertilization receptor, how to make embryo be successfully implanted into still
It it is so a key technology difficult problem facing of auxiliary procreation technology.When Embryonic limb bud cell, containing big in the microenvironment of implant site
The chemotactic factor of amount and Pro-inflammatory mediator.
Chemotactic factor is the signal protein of small molecule cytokine or emiocytosis, and the major function of chemotactic factor is
Participate in leucocyte migration.In reproductive medicine field, chemotactic factor rises emphatically in ovulation, menstruation, Embryonic limb bud cell and birth process
The effect wanted.
It is known that the chemotactic factor of Maternal-fetal interface is Embryonic limb bud cell must obtain the factor.There are some researches show, the uterus of people
Inner membrance there are 9 kinds of chemotactic factor height express: monocyte chemoattractant protein-3, eotaxin, ractalkine, huge
Phagocyte inflammatory protein-1 β, 6Ckine, IL-8, Hemofiltrate CC chemotactic factor-1, Hemofiltrate CC chemotactic because of
Son-4 and macrophage origin chemotactic factor.In uterus film is held phase and First Trimester, the up-regulated of chemokine mRNA.
Transplanting in early days, transplantation site has high-caliber Th1 type Pro-inflammatory mediator and an IL-6, IL-8, before TNF-α etc.
Inflammatory cytokine.The immunocyte that these Pro-inflammatory mediator are mainly raised by endometrial cell and transplantation site
Secretion.It practice, Embryonic limb bud cell process, placentation process, early pregnancy and midtrimester of pregnancy are in early days more like an open injury, this is open
Wound needs strong inflammatory response.At First Trimester, in order to implant, embryo must break through the epithelial layer in uterus, destroys uterus
Internal film tissue and invade, followed by, trophocyte replaces endometrium and parent blood vessel smooth muscle, in order to make Placenta Hominis fetus unit obtain
Obtain sufficient nutrition.In order to ensure fully repairing endometrium and clear cell debris, need an inflammatory environment.Therefore in early days
Gestation is properly termed as proinflammatory cell phase.
Recent studies have shown that, the successful plantation of embryo may be secondary to the inflammatory reaction of wound inducement；Embryonic limb bud cell portion
The chemotactic factor of position becomes positive correlation with Pro-inflammatory mediator level with the Clinical Outcome of IVF/ICSI patient；Embryonic limb bud cell position
Chemotactic factor and Pro-inflammatory mediator be the key that embryo successfully plants.
IL-8 is a member of chemotactic factor family, mainly by mononuclear cell, fibroblast, epithelial cell and endotheliocyte
Secretion.IL-8 has two main functions, and one is Chemotaxis Function, and two is to participate in the innate immune response relevant to inflammation, is referred to as
Pro-inflammatory mediator.
At whole menstrual cycle, IL-8 is primarily located within chamber epithelium, glandular epithelium and endotheliocyte；Before in uterus film is held
Phase and holding the phase, the expression of IL-8 is substantially increased.Because IL-8 is chemotactic factor, it is again Pro-inflammatory mediator, therefore at early pregnancy
With midtrimester of pregnancy in early days, the IL-8 of Maternal-fetal interface and implant site plays particularly important effect in terms of maintaining gestation.
In a word, our result of study shows, before transplanting, the IL-8 in embryo medium can detect.From shifting
Plant the IL-8 of front embryo as chemotactic factor and Pro-inflammatory mediator, may work at Embryonic limb bud cell position.Transplant forefathers
In embryo medium, IL-8 is positive, indicates pregnancy rate and Embryo Implantation Rate in that IVF/ICSI patient is higher, and the baby of patient lives
Product number increases, and indicates that IVF/ICSI patient may have high Clinical Pregnancy Rate in and relatively low abortion ratio.Human embryo training before transplanting
IL-8 in nutrient solution not only can improve the pregnancy rate of IVF/ICSI patient by the quality of prediction transplanting embryo, it is also possible to makees
It is that an independent index is to predict the Clinical Outcome of IVF/ICSI patient.IL-8 can comment before transplanting as patient IVF/ICSI
Index this method for valency fetal development potential combines existing morphological method and selects embryo to transplant, more existing
Alone morphological method select embryo transplant, pregnancy rate can improve more than 8-12%.The shape selected according to this method
High-quality transplanting embryo in state and high-quality and common transplanting embryo are transplanted to external fertilization receptor, then pregnancy rate is respectively
60.76% and 47.37%, such as the morphologic high-quality transplanting embryo not selected by this method and high-quality and common transplanting embryo
Transplant to IVF/ICSI patient, then pregnancy rate is respectively 52.59% and 34.85%.Existing morphology side is combined by this method
Method selects embryo to transplant, and more existing alone morphological method selects embryo to transplant, and pregnancy rate can improve 8-
More than 12%.
Claims (1)
1., by the method selecting the embryo that potentiality of development is good to improve test-tube baby Clinical Outcome, it is characterized in that this
Method comprises the following steps:
(1) on the same day transplanted, collect the cultivation before each embryo transfer of each external fertilization receptor and treat the training of transplanting embryo
Nutrient solution；First day of after fertilization in vitro, is confirming after fertilization, each germ cell of each external fertilization receptor is being placed respectively
In the spilting of an egg liquid drop of 30-50 μ L 10 % human serum albumin, put 37 DEG C, the incubator of 5% CO2 cultivated to embryo and move
Before planting；Embryo trained in vitro to the 3rd day, and the 4h-6h before carrying out embryo transfer, according to based on cleavage rates and morphology
Morphological assessment method is evaluated each external fertilization receptor and is treated the quality of transplanting embryo, and each external fertilization receptor transplants 2-3 every time
Individual embryo；Each external fertilization receptor wait transplant according to morphological assessment method embryo selected after, embryo to be transplanted is moved
Walk, i.e. collect to cultivate and treat the transplanting embryo spilting of an egg liquid drop containing 10% human serum albumin of 2 days, i.e. cultivate and treat transplanting embryo
Culture fluid；In view of each external fertilization receptor transplants 2-3 embryo every time, each embryo is both placed in one before transplantation
30-50 μ L cultivates 2 in the spilting of an egg liquid drop containing 10 % human serum albumins, each embryo transfer of each external fertilization receptor
Front cultivation treats that the culture fluid of transplanting embryo is to cultivate 2-3 respectively to treat the transplanting embryo ovum containing 10% human serum albumin of 2 days
Split liquid drop, cultivate this 2-3 the drop treating transplanting embryo before collecting each each embryo transfer of external fertilization receptor, form one
Part specimen, every a specimen is containing 25 μ L-40 μ L culture fluid；It is collected under similarity condition the cultivation not placing embryo cultivated
Liquid is as negative control；
(2) with liquid-phase chip high-throughout protein analysis technology (LHTPA) detect interleukin in every a specimen-
8, i.e. the content of IL-8；
(3) transplanting embryo is selected according to the content of interleukin 8 in morphological assessment method and specimen, the transplanting that will select
Embryo transfer is to external fertilization receptor；Available transplanting embryo morphology is improved quality same or similar, then select IL-in its culture fluid
8 is positive, and i.e. in culture fluid, IL-8 concentration range embryo between 0.19 pg/ml to 10.32 pg/ml transplants；
(4) if available transplanting embryo is improved quality to morphology differ or mutually the most similar, then select IL-8 sun in its culture fluid
Property, i.e. IL-8 concentration range embryo between 0.19 pg/ml to 10.32 pg/ml and/or select the form of the foetus state in culture fluid
The preferable embryo that improves quality transplants；
(5) time detecting IL-8 from making a collection of specimens to is 4h-6h, therefore i.e. can come by the method on the same day of embryo transfer
Screening the preferable embryo of potentiality of development transplant.
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|---|---|---|---|---|
| CN108982867A (en) * | 2018-05-31 | 2018-12-11 | 西北妇女儿童医院 | A method of quality of blastocysts is judged using protein chip |
| CN110361534A (en) * | 2018-03-26 | 2019-10-22 | 山大生殖研发中心有限公司 | It assesses embryo and predicts chemical markers and its application of success rate in vitro fertilization |
| CN110672857A (en) * | 2019-10-10 | 2020-01-10 | 四川大学华西第二医院 | TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110361534A (en) * | 2018-03-26 | 2019-10-22 | 山大生殖研发中心有限公司 | It assesses embryo and predicts chemical markers and its application of success rate in vitro fertilization |
| CN110361534B (en) * | 2018-03-26 | 2023-11-03 | 山大生殖研发中心有限公司 | Chemical markers for evaluating embryo and predicting success rate of in vitro fertilization and application thereof |
| CN108982867A (en) * | 2018-05-31 | 2018-12-11 | 西北妇女儿童医院 | A method of quality of blastocysts is judged using protein chip |
| CN110672857A (en) * | 2019-10-10 | 2020-01-10 | 四川大学华西第二医院 | TNF-alpha high-sensitivity Elisa detection kit and use method and application thereof |
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