Embryo and Implantation Disorders

Bolarinde Ola FRCOG MD

Outline

• Embryo and Embryo grading• Implantation physiology• Implantation failure • (Recurrent) Implantation failure• How to manage (recurrent ) implantation

failure?

Embryo Stages

Grades of (Day 3) Cleavage Stage Embryos:

Grade DescriptionGrade 1 Cells are of equal size; no fragmentation

seen

Grade 2 Cells are of equal size; minor fragmentation only

Grade 2.5 Cells are mostly of equal size; moderate fragmentation

Grade 3 Cells are of unequal size; no fragmentation to moderate fragmentation

Grade 4 Cells are of equal or unequal size; fragmentation is moderate to heavy

Blastocyst Grading

• Are graded 1 - 4 according to degree of expansion (and 5-6 degree of hatching)

• Blastocysts of grades 3 or higher are further graded A-C for Inner Cell Mass (ICM) and Trophectoderm

A - Tightly packed, many cellsB - Loosely packed, severalC - Very few cells

Blastocyst Stage Embryos: Day 5

.

BASIC IMPLANTATION PHYSIOLOGY

Embryo-Endometrium Communication

• Day 5 - Free floating blastocyst in utero • Day 6 - Blastocyst hatching – Start of Window• Day 7 - Blastocyst apposition to endometrium at

the beginning of the implantation window • Day 8 - Blastocyst adhesion occurring when LIF is

maximally expressed by the endometrium and blastocyst expresses the LIF receptor

• Day 9 - Blastocyst Invasion• Day 10 - Implantation complete– End of

Window

IMPLANTATION WINDOW AND EMBRYO-MATERNAL CROSSTALK

Justine S. Fitzgerald et al. Hum. Reprod. Update 2008;14:335-344

Free floating blastocyst

Blastocyst hatching

Blastocyst apposition

Blastocyst adhesion

Blastocyst invasion

Implantation complete

START (implantation

window)

END (implantation

window)

• Differentiation of trophoblast cells into inner cyto-trophoblast and outer syncytiotrophoblast layers and invasion into the luminal epithelium, where the blastocyst then commences to secrete cytokines such as IL-1, which in turn stimulates LIF expression in the endometrium

• Implantation is complete,• Implantation window is closed

REPRODUCTIVE FAILURE

• Embryo– Sperm factors– Egg factors

• Endometrium• Uterine• Tubal• Endocrine• Immune• Others

EMBRYO QUALITY

• Poor Sperm– Unexplained– Iatrogenic (surgery, radiation etc)– Chromosomal / Genetic– Congenital / varicocoele

• Reduced Ovarian reserve– Age– Iatrogenic– Chromosomal / Genetic– Congenital

ENDOMETRIUM

• Unexplained• Poor endometrial development• Infections• Polyps• Submucous fibroids• Congenital (Uterine Septa)

UTERINE / TUBAL

Uterine• Fibroids• Adenomyosis• Mullerian dysgenesis

Tubal• Hydrosalpinges

ENDOCRINE

• Hypothyroid• Hyperthyroid• Diabetes Mellitus• Polycystic Ovary Syndrome• Premature ovarian failure

IMMUNE DISORDERS

• Autoimmune thyroid disorders• Auto-immune gonadal diseases• Abnormal endometrial cytokines– Elevated NK cells in endometrium

• Antiphospholipid Syndrome - APS

IMPLANTATION

• Implantation is the series of stages including blastocyst hatching, adhesion, endometrial invasion, embryo differentiation and growth to produce a state of clinical pregnancy

• Measurable over a period by implantation rate:– Number of embryo implanted out of number transferred

over a period– i.e. total number of gestation sacs on scan divided by

total number of embryo transferred

FAILED IMPLANTATION

• Failure to reach a stage in which there is ultrasonographic evidence of intra uterine pregnancy (intra-uterine gestational sac)

IMPLANTATION FAILURE

Failure to achieve a clinical pregnancy • following the transfer of at least four embryos• at least 3 transfer cycles, fresh or frozen • in which embryos were good quality • in women aged <40 years

OUTCOMES OF IVF

• Ongoing clinical pregnancy• Cycle cancellation• Failed fertilisation• Failed implantation• Miscarriage• Ectopic pregnancy

• Implantation failure (repeated)

INVESTIGATIONS OF IMPLANTATION FAILURE

RECOMMENDED INVESTIGATIONS TESTS OF RESEARCH VALUE

• Ovarian reserve tests: FSH, AMH, antral follicle count

• Sperm DNA fragmentation

• Parental karyotype (?aneuploidies) • Natural Killer Cells

• Hereditable/acquired thrombophilia

• Pelvic imaging (TVS/TAS)

• Hysterosalpingography

• Hysteroscopy

• Laparoscopy

Improving the Embryo

IMPROVING EMBRYO QUALITY:GENERAL MEASURES

• Encourage couples to try early• Good diet• Reduce / Cigarette smoking• Reduce alcohol• Weight control

IMPROVING EMBRYO QUALITY, SPECIFIC MEASURES

• Sperm DNA fragmentation tests• Ovarian stimulation protocol• Gamete donation (for parental aneuploidies)• Insemination by ICSI• Careful embryo selection - embryoscope• Blastocyst transfer• Assisted hatching• Improved embryo transfer techniques

Improving the Endometrium

THROMBOPHILIA & IMMUNE DISORDERS

Antiphospholipid syndrome (APS)• A systemic autoimmune disease characterized by

vascular thrombosis and/or pregnancy morbidity in the persistent presence of antiphospholipid antibodies (aPL).

• aPL are currently evaluated by three tests – Abs against beta-2 glycoprotein I (anti-β2GPI abs) – Abs against cardiolipin (aCL), – Lupus anticoagulant (LA)

aPL cause defective placentation by interacting with both sides of the placenta. • At the decidual level, aPL is proinflammatory

with neutrophil infiltration, secretion of cytokines and complement activation

• At trophoblast level, aPL down-regulate hCG,

integrins and cadherins resulting in reduced trophoblast proliferation and growth

THROMBOPHILIA & IMMUNE DISORDERS

THROMBOPHILIA AND IMPLANTATION FAILURE

• APS is strongly linked to recurrent miscarriages (RCMs)

• Treatment of APS improves RCMs

• APS is NOT associated with implantation failure (IF)

• Treatment of APS does not usually improve IF

THYROID AUTOIMMUNITY

• Described as the presence of autoantibodies against thyroid peroxidase (TPO) and/or thyroglobulin (TG)

• Associated with repeated implantation failure (RIF)

• Treatment is Thyroxine supplements

NATURAL KILLER CELLS

• Natural killer (NK) cells are subpopulation of lymphocyte in peripheral blood

• Similar immune cells which are poor killers (uNK) populate the uterine lining at implantation – The functions of these uterine NK (uNK) cells are

essentially unknown – May have a role in regulating placentation and

trophoblast cell invasion

NATURAL KILLER CELLS

• Increasingly, clinicians are testing for uNK– No agreed range of normality

• An increasing number of treatments available– None conclusively proven to be beneficial

PERSISTENT ENDOMETRIAL FLUID

CAUSES OFFLUID WITHIN ENDOMETRIUM

• Ovarian stimulation (uterine or cervical)• High dose Estrogen• Low Estrogen• Tubal - Hydrosalpinges• Endometrial– Polyp or fibroid– Asherman’s synechia– Cervical stenosis– Chronic infection

• Others

POORLY DEVELOPED ENDOMETRIUM

POORLY DEVELOPED ENDOMETRIUM

• Poor ovarian reserve or POF• Iatrogenic (medical, radiation• Endometrial– Asherman’s synechia– Chronic infection

• Uterine fibroids• Adenomyosis• Others

THE ROLE OF DIAGNOSTIC HYSTEROSCOPY

Courtesies RCOG London StratOG

MANAGING IMPLANTATION FAILURE:Levels of evidence

Level 1+ : high quality meta-analyses of RCTs or RCT with a low risk of bias

Level 1- : meta analyses or RCTs or RCT with a high risk of bias

Level 2 : systematic review of case-control or well conducted case control cohort studies or cohort studies Level 3: case reports or case series

Level 4: expert opinion

LIVE BIRTH RATE PER CYCLE FOR ROUTINE HYSTEROSCOPY VERSUS NO

HYSTEROSCOPY PRIOR TO IVF/ICSI

Pundir & El Toukhi Reproductive BioMedicine Online 28, 2, 2014, 151–161

Level 2++ Evidence

PREGNANCY RATE: ROUTINE HYSTEROSCOPY VS NO HYSTEROSCOPY PRIOR TO IVF/ICSI (TWO PREVIOUS

FAILED IVF/ICSI

Bosteels, J. et al. Hum Reprod Update 2010 16:1-11

Level 1+ Evidence

ENDOMETRIAL POLYPS

Courtesies RCOG London StratOG

HYSTEROSCOPIC POLYPECTOMY VS DIAGNOSTIC HYSTEROSCOPY + BIOPSY

ONLY PRIOR TO IUI: CLINICAL PREGNANCY

Level 1+ Evidence

Bosteels J, Cochrane Database of Systematic Reviews 2013, Issue 1.

SUBMUCOUS FIBROIDS

Courtesy Mr B Ola FRCOG

HYSTEROSCOPIC MYOMECTOMY VS SEXUAL INTERCOURSE ONLY: CLINICAL PREGNANCY

Bosteels J, Cochrane Database of Systematic Reviews 2015, Issue 1.

Level 1- Evidence

UTERINE SEPTUM

Courtesies RCOG London StratOG; AND Mr B Ola FRCOG

UTERINE SEPTUM: TREATMENTLevel 2++ Evidence

Venetis 2015 Reproductive Biomedicine Online (Impact Factor: 3.02). 12/2014; 29(6):665-683.

UTERINE SYNECHIA

Courtesies RCOG London StratOG

HYSTEROSCOPIC SURGERY FOR ASHERMAN SYNECHIA:

Pregnancy and Live Birth Rates

Total Pregnant Live Births

43 51% 33%

A. Dawood, A. Al-Talib, T. Tulandi (2010). Obstet. Gynaecol. Can., 32 pp. 767–770

Evidence Level 2-

INTRAMURAL FIBROIDS

http://www.medicinenet.com/uterine_fibroids/article.htmhttp://www.advancedfertility.com/myomfoto.htm

EFFECT OF FIBROIDSLevel 2- Evidence

EFFECT OF MYOMECTOMY

Metwally M, Farquhar C, Li TC Cochrane Database of Systematic Reviews 2015, Issue 1.

Level 1- Evidence

ADENOMYOSIS

http://www.sir.net.au/adenomyosis

ADENOMYOSIS AND IVF CLINICAL PREGNANCY RATES

Level 2 Evidence

iVercellini P, et al. Uterine adenomyosis and in vitro fertilization outcome: a systematic review and meta-analysis. Human Reproduction. 2014 Mar 12:deu041.y

ADENOMYOSIS AND IVF MISCARRIAGESLevel 2 Evidence

iVercellini P, et al. Uterine adenomyosis and in vitro fertilization outcome: a systematic review and meta-analysis. Human Reproduction. 2014 Mar 12:deu041.y

ADENOMYOSIS AND IVF SHORT OR LONG PROTOCOL

Level 2 Evidence

iVercellini P, et al. Uterine adenomyosis and in vitro fertilization outcome: a systematic review and meta-analysis. Human Reproduction. 2014 Mar 12:deu041.y

HYDROSALPINGES AND TREATMENTS

Courtesy Mr B. Ola FRCOG

HYDROSALPINGES AND IVF

Salpingectomy prior to IVF in women with hydrosalingesimproves pregnancy, implantation and live birth ratesEvidence Level 1

Strandell et al 1999 Hydrosalpinx and IVF outcome : a prospective randomized multicentre trial in Scandinavia on salpingectomy prior to IVF Human Reprod 14:2762

Level 1+ Evidence

ENDOMETRIAL SCRATCH

Coughlan, C et al. Factors affecting the outcome of “Endometrial Scratch” in women with recurrent implantation failure. J Reprod Med. 2014.; 59 (1-2): 39 – 43

ENDOMETRIAL INJURY AND IMPLANTATION FAILURE

Carolina O Nastri Endometrial injury in women undergoing assisted reproductive techniques Cochrane Database of Systematic Reviews 2015, Issue 3

CONCLUSIONS

• Research is continuously active in the areas of embryo quality and implantation disorders

• There is still a big gap in knowledge

• Currently research is focussing on the roles of MicroRNAs in the regulation of cross-talk between embryo and endometrium