CN105911080B - A method of it is prominent quantitatively to enclose endometrium of implantation window stage pinocytosis - Google Patents
A method of it is prominent quantitatively to enclose endometrium of implantation window stage pinocytosis Download PDFInfo
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- 230000008884 pinocytosis Effects 0.000 title claims abstract description 99
- 210000004696 endometrium Anatomy 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000002513 implantation Methods 0.000 title claims abstract description 19
- 230000000007 visual effect Effects 0.000 claims description 56
- 238000005259 measurement Methods 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 230000002357 endometrial effect Effects 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 210000000981 epithelium Anatomy 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 5
- 238000013459 approach Methods 0.000 abstract description 2
- 206010003694 Atrophy Diseases 0.000 abstract 1
- 230000037444 atrophy Effects 0.000 abstract 1
- 210000001161 mammalian embryo Anatomy 0.000 description 15
- 238000005516 engineering process Methods 0.000 description 7
- 230000032692 embryo implantation Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000035935 pregnancy Effects 0.000 description 4
- 230000001850 reproductive effect Effects 0.000 description 4
- 210000000110 microvilli Anatomy 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 231100000535 infertility Toxicity 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 206010033266 Ovarian Hyperstimulation Syndrome Diseases 0.000 description 1
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical class C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000010429 evolutionary process Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 208000021267 infertility disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
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- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
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Abstract
The prominent method of endometrium of implantation window stage pinocytosis is quantitatively enclosed present invention finds a kind of, it will enclose the prominent scanning electron microscopic picture of endometrium of implantation window stage pinocytosis and combine with Image J software, record the prominent quantity of pinocytosis of different developmental morphologies respectively.This method mainly includes the hierarchical approaches (pinocytosis is prominent in development, and it is prominent prominent with atrophy phase pinocytosis to develop complete pinocytosis) and its scanned picture unique method for cell count in Image J software for three kinds of developmental morphologies that endometrium pinocytosis is dashed forward under scanning electron microscope.It, which is mainly solved, encloses the prominent quantitative problem of endometrium of implantation window stage pinocytosis.
Description
Technical field:
The present invention relates to a kind of qualitative assessments to enclose the prominent method of endometrium of implantation window stage pinocytosis, and one kind passing through endometrium
The prominent scanning electron microscopic observation of pinocytosis combines that the prominent method of endometrium pinocytosis is quantitatively evaluated with Image J software metering.
Background technique:
It is assessed according to the World Health Organization (WHO), there are about 1 couple of Mr. and Mrs, there are dysgenesias in every 7 couples of Mr. and Mrs.It adjusts in the recent period in China
It looks into, domestic infertility person accounts for the 10% of married couple's number, and 4.8% than investigation in 1984 doubles more, and disease incidence is presented
The trend of liter.Issues of Human Assisted Reproductive Technologies (Assisted Reproductive Technology, ART) can be controlled to a certain extent
Infertile couples are treated to achieve the purpose that fertility.It mainly includes artificial insemination, vitro fertilization-embryo implanting (In Vitro
Fertilization and Embryo Transfer, IVF-ET), intracytoplasmic sperm injection, science of heredity before embryo implantation
The technologies such as diagnosis, freezing of semen, embryo cryopreservation.Test-tube baby is exactly the baby given birth to using IVF-ET Technique
Youngster.Vitro fertilization-embryo implanting (in vitro fertilization-embryo transfer, IVF-ET) is also known as " first
For test-tube baby ", refer to and ovum and sperm are taken out out of human body respectively and are fertilized in vitro, after developing into embryo, then transplants
It returns in maternal uterine, to reach a kind of technology for purpose of becoming pregnant.
Assisted reproductive technology (ART) is grown rapidly in the past 30 years.Fertilization and embryo transfer (IVF-ET) in vitro
In, Controlled ovarian super-stimulation (COH) technology, the application of the technologies such as new Embryo Culture system and ultrasonic mediation embryo transfer, pole
The earth improves Clinical Pregnancy Rate in[1].However, Embryo implantation is unsuccessfully still a main problem in the treatment of infertility,
The Clinical Pregnancy Rate in of domestic and international many reproductive centers is hovered always 22.8%~40.5%[2,3].It is well known that embryo quality and
Endometrium receptivity is two principal elements for determining Embryo Implantation Rate in and Clinical Pregnancy Rate in.The acquisition of embryo quality and patient
The factors such as age, Ovarian hyperstimulation scheme and laboratory cultures system are related, when the acquisition of embryo quality reaches maintenance level, son
It is very important to Embryo implantation for Endometrium receptivity and inner membrance sexual factor synchronous with embryo's.
Psychoyos in 1976[4]It is put forward for the first time this concept of endometrium receptivity, i.e., endometrium allows embryo
The ability of bed, is a kind of state of specific period, also referred to as " implantation window " (WOI).Within this specific period, endometrium
Occur under Estrogen and progestin effect it is a series of adhere to embryo, invade relevant Morphology, can connect to greatest extent
It receives embryo, makes the smooth implantation of embryo, do not receive Embryo implantation too early or too late, that is, implantation window is closed.It is generally acknowledged that plantation
Window is open in midluteal phase, and optimal endometrium receptivity occurs approximately in greatly 6-8 days after the peak LH occurs[5,6], and occur special
The product of sign property.
In terms of cytomorphology, pinocytosis is prominent to be connect by most scholars as evaluation endometrium receptivity marker
By.It is the big and smooth membranaceous protrusion that endometrium of implantation window stage epithelial cell top occurs, most early in the uterus of mouse
It is found in inner membrance, later in people's implantation period
Also there are similar structures in 2 endometriums[7,8].The prominent evolutionary process of pinocytosis is by rank completely different in several forms
Section, therefore it can be divided into (developing in development；The D phase), develop completely (Fully Developed；The FD phase), decline
Phase (regressing；The R phase).The prominent cell volume of D phase pinocytosis starts to expand, and surface microvillus starts to attenuate, and shortens, and straightens；FD
Phase pinocytosis is dashed forward, and cell volume expansion is maximum, and such as " bubble sample ", surface is smooth, nothing or a little microvillus；The prominent surface of R phase pinocytosis occurs
A large amount of folds are similar to " ball for having let out gas ", and microvillus reappears.The pinocytosis of FD phase is prominent to be represented optimal endometrium and holds
Property.Although the prominent effect of pinocytosis is still unknown, they seem directly to take part in the process that blastocyst is adhered to endometrial surface.
Therefore, there may be potential clinical value with the prominent assessment endometrium receptivity of pinocytosis.However, some scholars think uterus
There is apparent heterogeneity in the prominent cell of inner membrance pinocytosis, be difficult to distinguish whole developmental morphology on developmental morphology and quantity
And quantity is quantified, so they, which think that pinocytosis is prominent, cannot objectively and accurately assess endometrium receptivity.
At present there are mainly two types of the prominent methods of assessment pinocytosis, a kind of method be the prominent developmental morphology of simple assessment pinocytosis and not
Quantity is carried out objectively quantitative[9]；3 prominent periods of pinocytosis are described in detail in this method, it is believed that FD phase born of the same parents in the picture visual field
Drink is prominent to be accounted for entire inner membrance percentage and is at best endometrium greater than 60% and hold the phase, and to the prominent number of the pinocytosis in each period
Amount is quantified, and objective quantitative approach is not provided yet.Another method is that everybody compares approval at present[10].This method
The developmental morphology and quantity progress subjective scoring dashed forward respectively to pinocytosis: D phase pinocytosis is prominent to be denoted as 1 point, and the FD phase is denoted as 3 points, and the R phase is denoted as
1 point；The prominent quantity of pinocytosis accounts for the percentage of the total endo cell number in the visual field and is denoted as 0 point for 0%, and 1%-20% is denoted as 1 point, 21%-
50% is denoted as 2 points, is denoted as 3 points greater than 50%.The total average mark of all pictures is finally acquired, average mark is high, then receptivity is opposite
Preferably.Above two method is comparatively more subjective, and roughly, variability is big, is unable to the prominent form hair of objective assessment pinocytosis
It educates and the expression of quantity, and then can accurately less assess endometrium receptivity.
Summary of the invention:
In order to solve the problems such as subjective larger, accuracy is poor in existing appraisal procedure, the present invention provides a kind of opposite
The prominent method of endometrium of implantation window stage pinocytosis is enclosed in objective assessment.
A kind of method quantitatively enclosing endometrium of implantation window stage pinocytosis and dashing forward of the present invention, includes the following steps:
Step 1. encloses endometrium of implantation window stage sample after treatment, by scanning electron microscope on endometrium
There are the visuals field that pinocytosis is dashed forward to take pictures for chrotoplast, and unified amplification factor × 2000 save picture with BMP format；
Picture is opened (File-open) by Image J software by step 2., then plug-in unit-analysis-cell count
(Plugins-Analyze-Cell Counter), Type 1 represent D phase pinocytosis and dash forward, and Type 2 represents FD phase pinocytosis and dashes forward, Type
3, which represent R phase pinocytosis, dashes forward；D phase, the quantity that FD phase and R phase pinocytosis are dashed forward, final result (Results) are in recordable picture respectively
The prominent quantity of three period pinocytosis is acquired respectively；
There are heterogeneous because of the prominent distribution there are three kinds of different developmental morphologies and on endometrium of pinocytosis for step 3.
Property, it at least needs to shoot so obtaining by the qualitative modeling (The Running Average Method) of continuous measurements
The N prominent visual field pictures of pinocytosis can be only achieved stable counting, and the specific operation method is as follows:
(a) draw a reference axis first, X-axis be it is quasi- count visual field number that the prominent scanning electron microscope of pinocytosis shoots (1,2,
3 ... ..N), Y-axis is that the prominent number of pinocytosis of FD phase accounts for the percentage of the prominent sum of pinocytosis, is indicated with FD%；
(b) the Y-axis FD% value in the n-th visual field is the 1st into the n-th visual field, and the prominent sum of full grown pinocytosis accounts for N number of
The percentage of the prominent sum of pinocytosis in the visual field；
(c) N number of visual field is counted, is the prominent shared pinocytosis of FD phase pinocytosis in the interior membrane sample with the obtained FD% in N number of visual field
Total percentage dash forward as base value；By the method for Running average, finding out can get makes a variation with FD% base value
Visual field number of the range within 5%；By the FD% in the 1st visual field, until the FD% value in the n-th visual field is drawn to axis of ordinates, with folding
Line connection；The region of FD% base value ± 5% is drawn, if being stepped up with visual field number, Lian Xu≤5 FD% value is located at the region
It is interior, indicate that, by counting this N number of visual field, FD% value obtained tends towards stability.
(d) we pass through the above method, when obtaining the visual field number N=60 that scanning electron microscope shooting pinocytosis is dashed forward, most of patient
FD phase pinocytosis dash forward quantity it is more stable.
The advantages of invention, is: objectively and accurately having evaluated the prominent quantity of pinocytosis of different developmental morphologies, is subsequent scientific research
Research and application provide a kind of reliable assessment endometrium receptivity method.In IVF-ET, plantation fails not repeatedly
Pregnant patient, main cause may be that endometrium receptivity is poor or endometrium is asynchronous with embryonic development, prominent with pinocytosis
Carry out evaluator's endometrium receptivity and embryo transfer time is adjusted according to the expression quantity that FD phase pinocytosis is dashed forward, is expected to improve
Clinical Pregnancy Rate in.In individualized treatment scheme, objective assessment pinocytosis burst method may have good clinical value.
Detailed description of the invention:
Fig. 1 is three kinds of prominent developmental morphology A of endometrium pinocytosis: pinocytosis is prominent in development；B: it is prominent to develop complete pinocytosis；C withers
Contracting phase pinocytosis is prominent.
Fig. 2 is the operating process schematic diagram of Image J software.
Fig. 3 is to measure to need how many views by the qualitative modeling (The running average) of continuous measurements
Open country can just obtain that the complete pinocytosis of stable development is prominent to be counted, a, b, c respectively represent three different sample FD phase pinocytosis dash forward it is stable
Picture visual field number, even if continuous 3 points initially enter gray area and tend towards stability in figure.From a figure as can be seen that from the 33rd
The visual field, FD phase pinocytosis is prominent to tend towards stability.In b figure, the 20th visual field FD phase pinocytosis is prominent to tend towards stability.In c figure, the 45th visual field FD
Phase pinocytosis is prominent to tend towards stability.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawing.
A kind of method quantitatively enclosing endometrium of implantation window stage pinocytosis and dashing forward of the present invention, includes the following steps:
1. enclose endometrium of implantation window stage sample after treatment, it is thin to endometrial epithelium by scanning electron microscope
There are the visuals field that pinocytosis is dashed forward to take pictures by born of the same parents, and unified amplification factor × 2000 save picture with BMP format.
2. picture is opened (File-open) by Image J software, then plug-in unit-analysis-cell count (Plugins-
Analyze-Cell Counter), Type 1 represents D phase pinocytosis and dashes forward, and Type 2 represents FD phase pinocytosis and dashes forward, and Type 3 represents the R phase
Pinocytosis is prominent.D phase, the quantity that FD phase and R phase pinocytosis are dashed forward are in recordable picture respectively, final result (Results) acquires respectively
The prominent quantity of three period pinocytosis.Such as Fig. 2.
3. because there is heterogeneous, institute in the prominent distribution there are three kinds of different developmental morphologies and on endometrium of pinocytosis
It is obtained with us by the qualitative modeling (The Running Average Method) of continuous measurements and at least needs to shoot
The prominent visual field picture of 60 pinocytosis can be only achieved stable counting, and Fig. 3 example, the specific operation method is as follows:
(1) draw a reference axis first, X-axis be it is quasi- count visual field number that the prominent scanning electron microscope of pinocytosis shoots (1,2,
3 ... ..60), Y-axis is that the prominent number of pinocytosis of FD phase accounts for the percentage (indicating with FD%) of the prominent sum of pinocytosis.
(2) the 1st visual field FD%, which are that the complete pinocytosis of development is prominent, accounts for the prominent percentage of the 1st all pinocytosis in the visual field.If there is 2
The visual field, the prominent sum of pinocytosis that the Y-axis FD% value in the 2nd visual field represents the FD phase in the 1st visual field and the 2nd visual field account for 2 visuals field
The percentage of the prominent sum of middle pinocytosis.And so on, the Y-axis FD% value in the n-th visual field is the 1st into the n-th visual field, has been developed
The prominent sum of full pinocytosis accounts for the percentage of the prominent sum of pinocytosis in N number of visual field.
(3) we have tentatively counted 60 visuals field, are FD phase born of the same parents in the interior membrane sample with 60 obtained FD% in the visual field
The percentage of the prominent shared prominent sum of pinocytosis of drink (as base value).It is anticipated that passing through the method for Running average, can look for
Can get out with FD% base value range of variation within 5% visual field number (it is desirable that this visual field number≤60, that is,
We can reach the level close to FD% base value by counting relatively little of visual field number).We are by the 1st visual field
FD% connected until the FD% value in the n-th visual field is drawn to axis of ordinates with broken line.The region of FD% base value ± 5% is drawn,
If being stepped up with visual field number, Lian Xu≤5 FD% value is located in the region, indicates by counting this N number of visual field, obtained
FD% value tends towards stability.
Claims (2)
1. a kind of quantitatively enclose the prominent method of endometrium of implantation window stage pinocytosis, include the following steps:
Step 1. encloses endometrium of implantation window stage sample after treatment, thin to endometrial epithelium by scanning electron microscope
There are the visuals field that pinocytosis is dashed forward to take pictures by born of the same parents, and unified amplification factor × 2000 save picture with BMP format；
Step 2. is opened picture by Image J software, then plug-in unit-analysis-cell count, and Type 1 represents D phase pinocytosis and dashes forward,
Type 2 represents FD phase pinocytosis and dashes forward, and it is prominent that Type 3 represents R phase pinocytosis；D phase, FD phase and R phase pinocytosis are in recordable picture respectively
Prominent quantity, final result acquire the prominent quantity of three period pinocytosis respectively；
Step 3. is because there is heterogeneous, institute in the prominent distribution there are three kinds of different developmental morphologies and on endometrium of pinocytosis
Show that at least needing to shoot the N prominent visual field pictures of pinocytosis can be only achieved stabilimeter with the qualitative modeling by continuous measurements
Number, the specific operation method is as follows:
(a) reference axis is drawn first, X-axis is to intend the visual field number of the prominent scanning electron microscope shooting of counting pinocytosis, 1,2,3,
... ..N, Y-axis are that the prominent number of pinocytosis of FD phase accounts for the percentage of the prominent sum of pinocytosis, are indicated with FD%；
(b) the Y-axis FD% value in the n-th visual field is the 1st into the n-th visual field, and the prominent sum of full grown pinocytosis accounts for N number of visual field
The percentage of the prominent sum of middle pinocytosis；
(c) N number of visual field is counted, is that the prominent shared pinocytosis of FD phase pinocytosis is prominent total in the interior membrane sample with the obtained FD% in N number of visual field
Several percentages are as base value；By qualitative modeling, finding out be can get with FD% base value range of variation within 5%
Visual field number；The FD% in the 1st visual field is connected until the FD% value in the n-th visual field is drawn to axis of ordinates with broken line；Draw FD%
The region of base value ± 5%, if being stepped up with visual field number, Lian Xu≤5 FD% value is located in the region, and expression passes through counting
This N number of visual field, FD% value obtained tend towards stability.
2. a kind of method quantitatively enclosing endometrium of implantation window stage pinocytosis and dashing forward as described in claim 1, it is characterised in that: scanning
The visual field number N=60 of Electronic Speculum shooting.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03285110A (en) * | 1990-03-31 | 1991-12-16 | Nec Home Electron Ltd | Measuring apparatus of angular velocity |
| CN103648497A (en) * | 2011-05-31 | 2014-03-19 | 金斯顿女王大学 | Methods and compositions for enhancing fertility and/or inhibiting pregnancy failure and restoring glucose tolerance |
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2016
- 2016-04-14 CN CN201610231009.2A patent/CN105911080B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03285110A (en) * | 1990-03-31 | 1991-12-16 | Nec Home Electron Ltd | Measuring apparatus of angular velocity |
| CN103648497A (en) * | 2011-05-31 | 2014-03-19 | 金斯顿女王大学 | Methods and compositions for enhancing fertility and/or inhibiting pregnancy failure and restoring glucose tolerance |
Non-Patent Citations (4)
| Title |
|---|
| AC型子宫内膜种植窗期胞饮突的形态学观察;李权等;《山东医药》;20081231;第48卷(第17期);第29-30页 |
| Fiji:an open-source platform for biological-image analysis;Johannes Schindelin等;《NATURE METHODS》;20120731;第9卷(第7期);第676-682页 |
| Image J在生物图像处理中的应用;张雯婷;《百度文库》;20160122;第1-5页 |
| 子宫内膜着床期血管生成状态及胞饮突表达对胚胎着床的影响;冯苗等;《中国实用妇科与产科杂志》;20100731;第26卷(第7期);第525-529页 |
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