Human Reproduction 2013 Jan;28(1):172-7 Presented by Hsing Chun Tsai

 EM < 7mm : suboptimal for embryo transfer and associated with reduced pregnancy chances  Proposed treatment remedies :  Extended estrogen if time allows  Low-dose aspirin  Pentoxifylline and tocopherol  Vaginal sildenafil citrate (Viagra ®)

 Prevalence (author’s experience) : < 1% IVF patients  Treatment challenges   cycle cancellations  unplanned cryopreservation of embryos  utilization of gestational carrier

Fertility and Sterility Vol. 95, No. 6, e13-e17, May 2011

 NCT : whether endometrial infusion with G- CSF, independent of endometrial thickness, in routine IVF cycles affects pregnancy rates?  NCT : whether, and to what degree, endometrial infusion with G-CSF, is able to expand chronically thin endometrium, which is resistant to standard therapies?

 Study design: prospective observational cohort pilot study over 18 months  Patients:  21 infertile women with endometrium < 7mm on the day of hCG administration in 1 st IVF cycle at this center  Previous tx including oral and vaginal ethinyl estradiol (E2: 2mg, per os bid and 1 mg per vagina tid) and sildenafil citrate (Viagra 25mg per vagina qid)

 Options offered: 1. Participation in a clinical trial 2. IVF cycle cancellation 3. Embryo transfer into an inadequately thin endometrium 4. Off-label use of G-CSF intrauterine infusion

 Ovulation induction with hCG 10,000 IU: ≧ 1 follicle ≧ 19 mm  Diagnosis of unresponsive thin endometrium : < 7mm by ultrasound on the day of hCG administration  G-CSF endometrial infusion: mcg/1ml (Nupogen™, Filgastrim ® ) approximately 6-12 h before hCG administration 2. Repeated G-CSF infusion if endometrium <7mm on TVOR day  3/21 cases (14.3%)  Endometrial thickness on ET day (universally Day 3)  Embryo transfer  EM ≧ 7mm

 Tomcat catheter  1-ml insulin syringe

 Primary outcome: ↑ endometrial thickness  Secondary outcome: clinical pregnancy rate  Statistical analysis: SPSS, version 18.0  p < 0.05  significant  Informed consent for “experimental” procedure

 All patients underwent embryo transfer (EM ≧ 7mm).  The clinical pregnancy rate was 19.1%. significant

 The present data are supportive of the initial report.  Small cohort size  Lack of a control group  0.1± 0.4 (range 0-1) of previously failed cycles  cancelled due to inadequately thin endometrium

 A growth spurt in endometrial thickness can be observed within 48h of G-CSF administration.  All but 3 pts reached a minimal thickness of 7mm < ~48h  How does G-CSF work? -- unknown

 Glycoprotein, growth factor and cytokine  Endometrium, macrophages and in other immunocytes  Therapeutic uses:  Stimulates the production of WBC  treat neutropenia (induced by chemotherapy or bone marrow transplant)  Increase the number of hematopoietic stem cells of the donor before collection  hematopoietic stem cell transplantation  CNS: inducing neurogenesis and anti-apoptosis ----> neurogenerative diseases  Enhancing endometrial receptivity ??  Filgrastim ® : recombinant human G-CSF synthesised in E. coli expression system

How to achieve rapid proliferation of endometrium ?

Production and physiological function of granulocyte colony-stimulating factor in non- pregnant human endometrial stromal cells. Tanaka et al., 2000 Department of Obstetrics and Gynecology, Osaka City University Medical School, Osaka, Japan  G-CSF enhances cAMP-mediated decidualization of human endometrial stromal cells in both an autocrine and a paracrine fashion.

Regulation of the cellular subpopulation ratios of normal human endometrial stromal cells by macrophage colony-stimulating factor Tanaka et al., 2003 Department of Obstetrics and Gynecology, Osaka City University Medical School, Osaka, Japan  M-CSF enhances G-CSF secretion from 8-Br-cAMP- unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells.  M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells.

Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract Fahey et al., 2005 Department of Physiology and Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, USA  G-CSF and GM-CSF are secreted apically in polarized epithelial cells.  The role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.

 G-CSF has been proposed as a treatment for implantation failure and repeated miscarriages. (immunological process)  G-CSF and GM-CSF: involved in reproductive functions  Cyclic changes of G-CSF mRNA in follicular fluid during MC cycle  G-CSF and its receptor in human luteinized granulosa cells  G-CSF in follicular fluid  useful biomarker of oocyte competence before fertilization (L édée et al., 2011)  M-CSF + HMG use in poor responders to improve follicle development (Takasaki et al., 2008)

 Expression of GM-CSF and its receptor during the menstrual cycle implies an autocrine and paracrine function of GM-CSF in the endometrium.  In the mice: GM-CSF suggested as an essential regulator in uterine dendritic cells during early pregnancy  Supplementation of embryo growth media with GM-CSF promotes human embryo development to blastocyst stage.  Embryo growth medium with GM-CSF became available commercially in Europe in 2011, and approved in USA in late 2012 (44% improvement in implantation rate)

 Synergistic effects between G-CSF and sildenafil citrate can not be ruled out.  How does G-CSF thicken the endometrium in such a short tome interval?  Dosage? frequency?  Route?  sc., intrauterine  Is M-CSF or GM-CSF better?  Beyond endometrial expansion, can G-CSF beneficially affect implantation and pregnancy rates in women undergoing IVF?

 Considering the characteristic of the patients (mean age: 40.5; diminished ovarian reserve: 76.2%; thin EM)  ongoing clinical pregnancy rate of 19.1% is remarkable  2011 ongoing clinical pregnancy rate for all women at age 41 in very similar population was 25%.  Natural conception rate in a general infertile population is estimated at ~1% per month.  Spontaneous pregnancy rate of at most, 3.6%, in 18-month period

 This pilot study supports the utility of G-CSF in the treatment of chronically thin endometrium.  G-CSF treatment will, in very adversely affected patients, result in low but very reasonable clinical pregnancy rates.