Tex gp technology talk by Fertility Associates

Matthew (Tex) VerMilyea, PhD, HCLD/CC

Scientific Director

Technologies

How to Choose the Best Embryo for Transfer? ² Observational Analysis: Embryo Development Morphology Assessment ² Genomic Analysis: Trophectoderm Cell Biopsy ² Transcriptomic / Metabolomic Analysis: Granulosa cells Cumulus cells Culture medium

TiMI

Embryo Development

One frame at a time

TimeLapse Morphometry Imaging

1-cell

cc1 t2: division to 2-cells t3: division to 3-cells t4: division to 4-cells t5: division to 5-cells t8: division to 8-cells

4-cells

2-cells

cc2

8-cells

cc3

cc1: duration of first cell cycle (cc1 = t2-t1) cc2: duration of second cell cycle (cc2 = t3-t2) cc3: duration of third cell cycle (cc3 = t5-t3) s2: duration of transition from two-blastomere embryo to four-blastomere (s2 = t4-t3) s3: duration of transition from five-blastomere embryo to eight-blastomere (s3 = t8-t5)

Photos courtesy of Matthew (Tex) VerMilyea, PhD, HCLD/CC Adapted from Meseguer et al., 2011

Discard?

DISCARD

(Morphology) INCLUDE

WITHIN RANGE

Exclude? OUTSIDE RANGE

s2 OUTSIDE RANGE

WITHIN RANGE

cc2 WITHIN RANGE

A+

A-

WITHIN RANGE

B+

OUTSIDE RANGE

WITHIN RANGE

cc2

cc2 OUTSIDE RANGE

EXCLUDE

OUTSIDE RANGE

B-

WITHIN RANGE

C+

cc2 OUTSIDE RANGE

C-

WITHIN RANGE

D+

OUTSIDE RANGE

D-

ORIGINAL ARTICLES: ASSISTED REPRODUCTION

Clinical validation of embryo culture and selection by morphokinetic analysis: a randomized, controlled trial of the EmbryoScope Irene Rubio, Ph.D.,a Arancha Gal! an, Ph.D.,a Zaloa Larreategui, Ph.D.,b Fernando Ayerdi, Ph.D.,b Jose Bellver, M.D.,a Javier Herrero, Ph.D.,a and Marcos Meseguer, Ph.D.a a

Instituto Universitario IVI Valencia, University of Valencia, Valencia; and b IVI Bilbao, Bilbao, Spain

Objective: To determine whether incubation in the integrated EmbryoScope time-lapse monitoring system (TMS) and selection supported by the use of a multivariable morphokinetic model improve reproductive outcomes in comparison with incubation in a standard incubator (SI) embryo culture and selection based exclusively on morphology. Design: Prospective, randomized, double-blinded, controlled study. Setting: University-affiliated private in vitro fertilization (IVF) clinic. Patient(s): Eight hundred forty-three infertile couples undergoing intracytoplasmic sperm injection (ICSI). Intervention(s): No patient intervention; embryos cultured in SI with development evaluated only by morphology (control group) and embryos cultured in TMS with embryo selection was based on a multivariable model (study group). Main Outcome Measure(s): Rates of embryo implantation, pregnancy, ongoing pregnancy (OPR), and early pregnancy loss. Result(s): Analyzing per treated cycle, the ongoing pregnancy rate was statistically significantly increased 51.4% (95% CI, 46.7–56.0) for the TMS group compared with 41.7% (95% CI, 36.9–46.5) for the SI group. For pregnancy rate, differences were not statistically significant at 61.6% (95% CI, 56.9–66.0) versus 56.3% (95% CI, 51.4–61.0). The results per transfer were similar: statistically significant differences in ongoing pregnancy rate of 54.5% (95% CI, 49.6–59.2) versus 45.3% (95% CI, 40.3–50.4) and not statistically significant for pregnancy rate at 65.2% (95% CI, 60.6–69.8) versus 61.1% (95% CI, 56.2–66.1). Early pregnancy loss was statistically significantly decreased for the TMS group with 16.6% (95% CI, 12.6–21.4) versus 25.8% (95% CI, 20.6–31.9). The implantation rate was statistically significantly increased at 44.9% (95% CI, 41.4–48.4) versus 37.1% (95% CI, 33.6–40.7). Conclusion(s): The strategy of culturing and selecting embryos in the integrated EmbryoScope time-lapse monitoring system improves reproductive outcomes. Clinical Trial Registration Number: NCT01549262. (Fertil Steril! 2014;102:1287–94. "2014 Use your smartphone by American Society for Reproductive Medicine.) to scan this QR code Key Words: Early pregnancy loss, embryo culture, embryo selection, implantation, ongoing and connect to the pregnancy rate, time-lapse Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/rubioi-embryo-culture-selection-morphokinetic-analysis/

n recent years, clinical practice efforts have been directed toward improving embryo selection. The

identiﬁcation of embryos with a higher capacity for implantation means we can reduce the number of embryos for

Received January 17, 2014; revised and accepted July 9, 2014; published online September 11, 2014. I.R. has nothing to disclose. A.G. has nothing to disclose. Z.L. has nothing to disclose. F.A. has nothing to disclose. J.B. has nothing to disclose. J.H. has nothing to disclose. M.M. has received payment for lectures from Ferring and Merck Serono. The instrumentation, disposables, and utensils used in this study were fully paid for by IVI. IVI is a minor shareholder in UnisenseFertiliTech A/S, but none of the authors have any economic afﬁliation with UnisenseFertiliTech A/S. Reprint requests: Marcos Meseguer, Ph.D., Instituto Valenciano de Infertilidad, Plaza de la Policía Local, 3, Valencia 46015, Spain (E-mail: marcos.meseguer@ivi.es). Fertility and Sterility® Vol. 102, No. 5, November 2014 0015-0282/$36.00 Copyright ©2014 The Authors. Published by Elsevier Inc. on behalf of the American Society for Reproductive Medicine. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/3.0/). http://dx.doi.org/10.1016/j.fertnstert.2014.07.738 VOL. 102 NO. 5 / NOVEMBER 2014

discussion forum for this article now.*

* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.

transfer without reducing the chances of pregnancy in a cycle of assisted reproduction. To this end, different noninvasive embryo selection methods have been designed that provide information on how to distinguish embryos with better prognosis (1) There are different methods of embryo gradation (2), but they are all based on morphology, and evaluation of morphology under microscope is subject to observer subjectivity (1). One of the noninvasive embryo evaluation methods to have come into the limelight in recent years is the time-lapse 1287

•  Time-Lapse and multivariable morphokinetic classification tree improves outcomes compared to standard incubators and traditional morphology assessment. •  Ongoing pregnancy rate increased from 41.7% to 51.4% (95% CI P-value .005) •  Early pregnancy loss decreased from 25.8% to 16.6% (95% CI P-value .01) •  Implantation rate increased from 37.1% to 44.9% (95% CI P-value .02)

Reproductive BioMedicine Online (2014) 29, 729–736

w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m

ARTICLE

Computer-automated time-lapse analysis results correlate with embryo implantation and clinical pregnancy: A blinded, multicentre study Matthew D VerMilyea a, Lei Tan b, Joshua T Anthony a, Joe Conaghan c, Kristen Ivani d, Marina Gvakharia e, Robert Boostanfar f, Valerie L Baker g, Vaishali Suraj b, Alice A Chen b, Monica Mainigi a, Christos Coutifaris a, Shehua Shen b,* a Penn Fertility Care, University of Pennsylvania, 3701 Market St. Suite #800, Philadelphia, PA 19104, USA; b Auxogyn, Inc, 1490 O’Brien Drive, Suite A, Menlo Park, CA 94025, USA; c Pacific Fertility Center, 55 Francisco Street, Fifth Floor, San Francisco, CA 94133, USA; d Reproductive Science Center of the Bay Area, 3160 Crow Canyon Road, San Ramon, CA 94583, USA; e Palo Alto Medical Foundation Fertility, Fertility Physicians of Northern California, 2581 Samaritan Drive, San Jose, CA 95124, USA; f HRC Fertility, 15503 Ventura Blvd, Suite 200, Encino, CA 91436, USA; g Stanford Fertility & Reproductive Medicine Center, 900 Welch Road, Suite 350, Palo Alto, CA 94304, USA

* Corresponding author. E-mail address: sshen@auxogyn.com (S Shen). Matthew VerMilyea, PhD, HCLD, is Director of Assisted Reproductive Technologies and Andrology Laboratories at the University of Pennsylvania, USA, and Scientific Director of Fertility Associates, New Zealand. He graduated from Cornell University, and received training in human embryology at Shady Grove Fertility, USA. He then obtained his PhD in Epigenetics from the University of Birmingham, UK. Matthew received a postdoctoral fellowship from the Japanese Society for the Promotion of Science for mammalian molecular embryology research at the RIKEN Institute, Kobe. Research interests include non-invasive and uninterrupted embryo culture protocols, time-lapse video imagery, micro-environment culture settings and early embryo histone modifications.

Abstract Computer-automated time-lapse analysis has been shown to improve embryo selection by providing quantitative and ob-

jective information to supplement traditional morphology. In this multi-centre study, the relationship between such computerderived outputs (High, Medium, Low scores), embryo implantation and clinical pregnancy were examined. Data were collected from six clinics, including 205 patients whose embryos were imaged by the EevaTM System. The Eeva scores were blinded and not considered during embryo selection. Embryos with High and Medium scores had significantly higher implantation rates than those with Low scores (37% and 35% versus 15%; P < 0.0001; P = 0.0004). Similar trends in implantation rates were observed in different IVF centres each using their own protocols. Further analysis revealed that patients with at least one High embryo transferred had significantly higher clinical pregnancy rates than those with only Low embryos transferred (51% versus 34%; P = 0.02), although patients’ clinical characteristics across groups were comparable. These data, together with previous research and clinical studies, confirm

Known Implantation 40% 30%

37% (41/111)

p = 0.003

20% 23% (50/220)

10% 0%

High Eeva High

Low Eeva Low

Multi-well Eeva™ Dish provides individual culture within the same media drop

Eeva Test results provide objective information to assist embryo selection

Eeva System using /me lapse imaging and intelligent computer vision so6ware collects data inside a standard incubator

Using a proprietary algorithm, images are automatically analyzed

How to Choose the Best Sperm for Fertilisation? WHO ‘Normal Range’ Semen Volume

1.5 ml or more

Sperm Concentration

15 million /ml or more

Proportion of sperm motile

40% or more

PICSI IMSI Proportion& with progressive motility Choosing the Proportion of sperm with normal shape, using strict best criteria sperm •  •

32% or more 4% or more

There are several ways to ensure that you are getting the best chance of pregnancy in IVF treatment. This fact sheet summarises the techniques available to help sperm selection in IVF-related treatment.

Conventional IVF: ~100,000 sperm added to each egg Mature sperm bind to a substance called hyaluronic acid (HA) as they make their way through the cells protecting the egg in the human Fallopian tube. This is one of several mechanisms the body is known to use to select a good sperm to fertilise the egg.

ICSI : Poor movement, number and shape ICSI with PICSI • Washed sperm are tested to see what

PICSI may improve pregnancy rates

Conventional In-Vitro Fertilisation

Intracytoplasmic Sperm Injection (ICSI)

Intracytoplasmic Morphologically selected Intracytoplasmic Sperm Injection (ICSI) Sperm Injection (IMSI)

x100 Magnification

x600-6,000 Magnification

Knez et al. Reproductive Biology and Endocrinology 2011, 9:123 http://www.rbej.com/content/9/1/123

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•  Sperm do not have efficient DNA repair mechanisms. •  Round vacuoles identified in sperm heads are associated with DNA fragmentation.

Figure 1 Classification of spermatozoa selected at 6,000 × magnification into 3 different categories. Class I - spermatozoa of good quality, Class II - spermatozoa of worse quality, and Class III - spermatozoa of poor quality. Legend: a,b,c - spermatozoa of Class I; d,e,f spermatozoa of Class II; g,h,i - spermatozoa of Class III.

•  DNA damage caused by Reactive Oxygen Species (ROS) can be associated with age, temp, varicocele, smoking, diet, environmental toxins.

•  No implantation after >4 embryos transferred.

embryos at the blastocyst stage were transferred into the uterus on day 5 by the TDT catheter set (CCD, Neuilly, France); if blastocysts did not develop, one or two morula were transfered, whereas lower-cell embryos were not transfered. Biochemical pregnancy was confirmed by the positive serum b-hCG test (> 48 mIU/ml) 15 days after the embryo transfer, and clinical pregnancy by an ultrasound scan of the gestational sac and the embryo heart beats 14 days after the positive b-hCG test.

SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) statistical program for Microsoft Windows was used for statistical calculations. The laboratory outcomes were expressed as fertilization rate (number of fertilized oocytes per injected oocytes), number of blastocysts (%), number of blaststocysts per cycle, and number of cycles with at least one blastocyst (%). The clinical outcomes were expressed as implantation rate (number of implanted embryos per transferred embryos), number of pregnancies, pregnancy rate per cycle, and number of spontanous abortions. For comparing categorical data, the chi-square (c2) test was performed, and Spearman’s rank correlation coefficient was used as a non-parametric measure of statistical dependence between the two variables.

•  Poor embryo development from Day3.

Statistics

To evaluate the role of sperm selection by their morphology, the laboratory and clinical outcomes between the IMSI and ICSI groups of couples were compared. The

•  Recurrent miscarriage from natural miscarriage.

•  Fertility Associates is committed to providing the latest technology to our patients. •  Time Lapse Morphometry Imaging (TiMI) service now available at FA which provides an uninterrupted growth environment and detailed assessment of embryological milestones. •  Intracytoplasmic Morphologically selected Sperm Injection (IMSI) by Ultra-high magnification selection is available and can improve reproductive outcomes.

Matthew (Tex) VerMilyea, PhD, HCLD/CC

Scientific Director

One Healthy BabyTechnologies at a Time.

Thanks for the opportunity!