Embryo grading is important in IVF to select good quality embryos for transfer based on developmental rate and morphology. The most followed grading systems are Gardner and Istanbul consensus, which assess embryos daily from fertilization to blastocyst stage based on criteria like cell number, size, and fragmentation. Good quality embryos with early cleavage and cell number on day 2 often develop into good blastocysts. Donor oocyte and sperm criteria and screening are also outlined to follow regulations. Oocyte donors can donate up to 7 oocytes only once in their lifetime from age 23-35.

1. Embryo Grading
and ART
summary
Kajal Patel
Junior Embryologist
Crysta fertility and IVF centre,
Pune, India

2. Importance
of embryo
grading
 Embryo Grading is a debatable topic in IVF since long.
 Embryos are selected for transfer on the basis of
developmental rate and morphological features under
the microscope.
 A good quality embryo increase the chances of
pregnancy rate and decrease the number of embryos
transferred.
 Most followed embryo grading system in the world
are:
1) Gardner grading system (assessment as per embryo
development day).
2) Istanbul consensus 2011 workshop on embryo
assessment (assessment as per embryo development
every hour).

3. Sperms and Oocytes
 Spermatozoa is a male reproductive cell or a gamete
and consist of a head, neck, tail and midpiece.
 Normal sperm have the following characteristics:
 The head is 5-6 µm long and 2-3 µm width.
 Midpiece is 5µm and tail is 50µm in length.
 Total size of the normal sperm is 60µm.
 Sperm morphology is depend on head, tail,
neck(0.1µm) and midpiece.
 The normal morphology range for spermatozoa is
=>4%.
 An Oocyte is a female gamete spherical in shape and
120µm in size.
 Oocyte maturation stages are:
 GV (Germinal vesicle)
 MI (Immature)
 MII (Mature Oocyte)
 Development of good embryo is directly related to
normality of nuclear and cytoplasmic maturation of
oocyte during preovulatory period.
 All anomalies should be divided into two categories due
to desynchronization of nuclear and cytoplasmic
maturation
1) Intra-cytoplasmic: refractile bodies, dense central
granulation, vacuoles, SER, etc.
2) Extra-cytoplasmic: polar body morphology, perivitelline
space(PVS), size and granularity, discolouration, defects in
zona pellucida (ZP), shape anomalies, etc.

5. Schematic drawings of some abnormal forms of human
spermatozoa

6. Oocyte Structure

7. a) Mature oocyte (MII)
b) Immature germinal vesicle breakdown (GVBD) oocyte (M1)
c) Immature germinal vesicle (GV)

8. Normal and
abnormal
Oocyte

9. Selection Criteria of an embryo
Morphology:
Number of cells, shape, size, symmetry and other factors like pro-
nucleate oocyte morphology, 2 cell stage to blastocyst stage of an
embryos.
Time: Embryo development as per the normal cell cycle.

10. Assessment of Fertilization (Day-1
17±1hr post-insemination)
 A fertilized oocyte should have two pronuclei and two polar bodies.
 Zygote arising from conventional IVF are observed 2hr behind those of
ICSI.
 Assessment for fertilization is usually done 17±1 hr post-insemination.
 Syngamy is seen around 20±1hr post-insemination and first cleavage by
24 hr.
 Pronuclear scoring takes into account the symmetry and alignment of the
pronuclei, the number and the position of NPBs( z score).

11.  The pronuclei are of similar size, closely
apposed and centrally located in the
fertilized oocyte, should have 3-7 NBPs in
each pronucleus.
 If any change in quality of number or
distribution of the NBPs within the
pronuclei it is consider to be abnormal.

12. Assessing cleavage-stage
embryo(day2 44±1hr post-insemination)
 Cell number: Embryos that have cleaved more slowly or faster than expected rate that have
reduced implantation(i.e. 2 cell or more than 6 cell on day2 or 44±1hr)
 Normally a day 2 embryo or 44 hours embryo is a 4-6 cells.
 Fragmentation: Anuclear, membrane bound extra cellular cytoplasmic structure. It can be
differ from cell by diameter on day2 <45µm (10%-mild, 10-25%-Moderate, >25%-severe).
 Multi-nucleation: The presence of more than one nucleus in one blastomere and micronuclei.
It should be performed on day2(44±1 hr post-insemination). Culture media, improper
temperature control during oocyte retrieval, stimulation of high oestradiol levels are
responsible factors for it.
 Cell size: Symmetrical and blastomere should be even sized.
 Other morphological features: Cytoplasmic granularity, membrane appearance and presence
of vacuoles.

13. Fragmentation

14. Cleavage stage
Day1: 24±1 hr. post-insemination
Day2: 44±1 hr. post-insemination
Cell number
Equal-size of cells
Cell nuclei
Cell fragmentation: <45µm

15. Assessment of Day3 68±1hr post-
insemination
 Cell number
 Equal-size of cells
 Cell nuclei
 Cell fragmentation: size<40µm.
 Slow embryo: <6 cells on day3(68±1 hr. post-
insemination)
 Accelerated: >9 cells embryo on day3(68±1 hr.
post-insemination)

16. Gardner grading system

17. Istanbuconsensus
 Istanbul consensus
Istanbul consensus
Istanbul consensus

18. Day 4 assessment(Morula
stage) 92±2hr Post-
insemination
 Embryo at this stage(92±2 hr.) would be
compacted or compacting and cell
boundaries become less visible.
 The day 4 scoring strategy is broadly not
been accepted but it provide single embryo
transfer rates similar to day5 single-embryo
transfers.
 The morula stage is the final stage prior to
formation of a fluid filled cavity called the
blastocoel cavity.

19. Blastocyst stage of an embryo Day 5
116±2hr post-insemination
 Blastocyst grading is based upon:
a) Expansion
b) Inner cell mass(ICM)
c) Trophectoderm cells(TE)
ICM: is significant for the development of the fetus itself.
TE: is the embryo's ability to attach and implant in the endometrium.
The fluid-filled cavity, which will form a structure called the yolk sac.

20. Blastocyst
Image

21. Early blastocyst
 4 to 5 days or 105 hr after retrieval, the embryo begins to differentiate into two
different types of cell: ICM and TE
 Once the cavitation has occurred, we can see the fluid in the cavity between the
cells and we call the embryo an early blastocyst.
 If a blastocyst is collapsed at the time of assessment, the blastocyst should be re-
evaluated 1-2 hr later as regular cycles of collapse and re-expansion of blastocyst
is normal.

22. Comparison-Istanbul and Gardner

23. Blastocyst development

25. Blastocysts
Images

26. Summary
 The Most followed techniques for embryo grading are Gardner and Istanbul
consensus 2011.
 The Embryos should be checked on following days:
Fertilization: Day 1 17±1 hr. post-insemination
Cleavage stage: Day1 24±1 hr. post-insemination
Day2 44±1 hr. post-insemination
Day3 68±1 hr. post-insemination
 Morula stage: Day4 92±2 hr. post-insemination
 Blastocyst stage: Day5 116±2 hr. post-insemination
 Some useful points for comparison and prediction of Blastocyst:
(1) Strict time of an assessment.
(2) Early cleavage and cell number on day 2(44±1hr post-insemination) are useful for predict
the development of a good-morphology blastocyst on day5.

27. Assisted Reproductive Technology-ART
act 2022
Total 16 Gazettes got Published till the date.
• The ART Regulation Act 2021 was first published on 20th December
2021 by Department of Law and Justice as per Section 42, later Rules
got published as Gazette on 7th June named as ART Rule 2022.
• Rules highlight about the procedures in details.
• In ART Surrogacy Act and ART Surrogacy Rule, there are few differences
like Act is above Rule but Rule is enacted by sections of Act only.
• If anything, which was not published as Gazette, has actually no Legal
value.
• Hence need to be carefully read and implemented in practice.

28. Assisted reproductive clinics-Level 1
 Min Staff: 01 Gynaecologist – Should be medical post graduate in
gynaecology and obstetrics.
 Registration fees: 50,000 INR for 5 yrs.
 Equipment & Infrastructures: (i) Microscope, (ii) Centrifuge, (iii) Refrigerator
 No mention about size / shape / orientation of sterile and Non-sterile zone.
 Do's: Can perform OPD, Folliculometry by USG, Semen Preparation and IUI
(including Donor IUI), husband sperm freezing for back up.
 Don't: Cannot perform Donor Gamete Cryopreservation or any ART Bank related
work, IVF, ICSI, ET, PGT, Surrogacy and Research are prohibited.

29. ART Clinics-Level 2
 Registration fees: 2,00,000 INR for 5 yrs.
 Min Staff:
1) Gynaecologist, 1 Anaesthetist, 1 embryologist and 1 Counsellor, in additional staff
at the level of Director and Andrologist may be employed but not mandatory.
 Qualification:
1) Gynaecologist-PG in OG with at least 50 OPU along with 3 yrs. experience in
Infertility or MD or FNB with at least 3 yrs. Experience.
2) Anaesthetist: Anaesthetist will be a medical post-graduate in Anaesthesia.
3) Counsellor: A person who is a graduate in Psychology or Clinical Psychology or
Nursing or Life Sciences.
4) Director: The director shall have a post-graduate degree in Medical or Life
Sciences or Management Sciences.

30. 5) Andrologist: The Andrologist in a clinic or a bank will be a Mch or DNB in Urology with
special training in Diagnosing and Treating in Male infertility.
(6) Embryologist: Full time MCE with 3 yrs. human ART laboratory experiences in
handling human gametes and embryos; Full Time Ph. D (Thesis related to Clinical
Embryology or ART or fertility) with additional one year of human ART laboratory
experience in handling human gametes and embryos; MBBS or BVSc should have MCE
(full time) with 2 yrs. ART laboratory experience in handling human gametes and
embryos; MSc in Biotech or Life Sciences minimum of one year of on-site, fulltime
clinical embryology certified training in addition to four yrs. experience in
handling human gametes and embryos in a registered ART level 2 clinic.

31.  Equipment & Infrastructures: (a) Microscope; (b) Incubator (minimum 02 in number); (c)
Laminar Airflow; (d) Sperm counting Chambers;(e) Centrifuge; (f) Refrigerator; (g) Equipment
for cryopreservation; (h) Ovum Aspiration Pump; (i) USG machine with transvaginal probe and
needle guard; (j) Test tube warmer and (k) Anaesthesia resuscitation trolley.
 No mention about size/shape /physical requirement/ orientation of sterile and non-sterile
zone.
 Do's: Can perform OPD, Folliculometry by USG, IUI, IVF,ICSI, PGT, Any Surgical Sperm
Extraction Procedure i.e. TESA, PESA, TESE and Research. They can perform any ART
procedure including Donor IUI/IVF. Donor Oocyte retrieval, post Controlled ovarian
Stimulation and up to 7 oocytes can be retrieved.
 Cryopreservation Facility: Donor Oocyte Freezing, Husband Sperm Freezing for back up,
Wife’s Oocytes, Freezing for back up, ‘Social’ Egg or Sperm Freezing can be done for future
self-use, Minor’s or Adult’s Sperm/Oocytes/Testicular / Ovarian tissue Freezing can be done
before any Malignancy treatment or Procedure and can be used for self-ART Cycle, in future
post recovery from the disease as per patient’s wish.

32. Some
Importance
points for
Oocyte(23-
35 Yrs.) &
Sperm
Donation(21
-55 Yrs.)
 Any Women(23-35 Years) irrespective of her marital status, or history
of pregnancy and/or motherhood can become an Oocyte Donor and
should be free from STDs or Genetical disease and/ medically fit.
 A bank shall not supply the sperm or oocyte of a single donor to more
than one commissioning couple (Section 27, Sub Section 3). An
oocyte donor shall donate oocytes only once in her life and not more
than seven oocyte shall be retrieved from the oocyte donor.
 Interpretation of these two Sub Sections of same section 27 is, i) ART
Bank can supply donated Sperm of a particular Sperm Donor only
once to a particular ART Clinic (Level 1/2) for a particular patient.
Nowhere it is mentioned whether Male can Donate only once in Life.
So there is a possibility of interpretation in such way that One Sperm
Donor sample may be supplied once to a single clinic for a single
patient and for as many time as required if pregnancy doesn’t take
place to that particular couple (if that couple wants to use that sperm
donor sample only), but can be supplied to other ART Clinics for
other intending couples but cannot use multiple times for different
patients of a particular ART Clinic .

33. ii) Oocyte Donor can donate only 7 oocytes, either in ART Clinic or Bank, only once in her Lifetime.
Controlled ovarian stimulation to be practiced carefully to avoid OHSS.
 Oocyte Donor information should be kept strictly confidential between 5 parties viz. i)
Oocyte Donor, ii) ART Clinic authorized personnel and iii) ART bank authorized personnel iv)
Commissioning Couple and v) National Registry database only.
 Oocyte Donation for any commercial gain is strictly prohibited under the ART Regulation Act
2021.Donors must be insured by IRDA registered General Health Insurance company (Check in
www.irdai.gov.in), of sufficient amount, before Ovum Pickup procedure, for 12 months and
that to be purchased by Commissioning Couples and handover to the Donor.
 The Commissioning couple/woman shall sign an affidavit to be sworn before Metropolitan
Magistrate or a Judicial Magistrate of First Class or an Executive Magistrate or a Notary
Public giving guarantee as per the section 22 (4)(ii) of the Assisted Reproductive Technology
(Regulation) Act, 2021 [ART Act 2021: 22 (4) (ii) : “insurance” means an arrangement by which
a company, individual or commissioning couple undertake to provide a guarantee of
compensation for specified loss, damage, complication or death of oocyte donor during the
process of oocyte retrieval;].

34.  Donor must be screened for HIV Type 1 and 2, HBV, HCV and Treponema pallidum (syphilis)
through VDRL (As per ART Regulation Rule 2022, Section 10) along with Blood Group, Diabetes and
Thalassemia.
 Unused Donor Gametes or Embryos can be frozen and kept in Cryo facility for maximum of 10
years and later on can donate it for research with the sign of commissioning couple or individual.
 The sale, transfer or use of gametes, zygotes and embryos, or any part thereof or information
related thereto, directly or indirectly to any party within or outside India shall be prohibited
except in the case of transfer of own gametes and embryos for personal use with the permission
of the National Board (Section 29).
 Insurance amount is not mentioned anywhere, only few companies are offering 1 lac as max and 2
lac is min amount. till date, no General Health Insurance company, on papers, are promising to
neither cover any Oocyte Retrieval related complications nor they promise to cover Ovarian
Hyperstimulation Syndrome from Day 1 of Insurance Purchase).
 This Act and Rule ensures the safety of Oocyte Donor’s health (Insurance + Affidavit), by
introducing revolutionary Section 12(i) & (ii) of ART (Regulation) Rule 2022 which is powered by
Section 22 (4) (ii) of the ART (Regulation) Act, 2021.

35.  Don'ts: Level 2 ART Clinic cannot perform Donor Sperm Cryopreservation. They
also cannot offer Surrogacy related any services if they don’t have Surrogacy
Registration along with ART Level 2 Reg.
 Some consents form should be maintain such as the couple or woman as per
specified in Form-6, IUI-husband semen(form 7), IUI-donor semen(form
8), freezing of embryos(form 9), for freezing gametes(form 10), assent for
freezing of gametes sperm or oocytes and parental consent(form 11), oocyte
retrieval(form 12), oocyte donor(form 13).

36. ART banks
 Registration fees: 50,000 INR for 5 yrs.
 Min staff: 1 Registered Medical Practitioner (trained in the
handling, preparation and storage of Semen samples).
 Equipment & Infrastructures:(a) Centrifuge
machine;(b)Incubator;(c) Microscope and (d) Laminar Air Flow.
 No mention about size/shape / physical requirement /
orientation of sterile and Non sterile zone.
 Do's: Male and Female Donor Registration, Semen analysis of
Donor, Blood test of Donor, Cryopreservation of Donor Sperm,
optionally they can have Donor Oocyte Freezing and maintained all
the records.

37.  Don’ts: They can neither stimulate any Patients and perform any ART Procedure
such as Male/Female Gamete retrieval and freezing of Sperm/Oocytes of those
patients, nor they can provide any ART Treatment such as IUI, IVF, ICSI, ET or
Surrogacy to any Patient.
 The Art banks shall maintain following forms: (i) record of use of donor gametes as
specified in Forms 14,14 A and 14B;
 (ii) consent form for the donor of sperm as specified in Form 15.

38. Surrogacy Clinic:
 Registration fees: 2,00,000 INR for 3 yrs.
 Staff Requirement & their Qualification, Equipment and Infrastructures are all same
as ART Level 2 Clinic.
Steps For Intending Couple/ Intending Women of Indian Origin To Avail Surrogacy
Under The Surrogacy (Regulation) Act, 2021.
1.Certificate of Recommendation-a couple of Indian origin or an intending woman
who intends to avail surrogacy, shall obtain a ‘Certificate of Recommendation’ from the
National Board*. Apply in Form 1 with self-attested required documents. [see Rule
4, the Surrogacy Rule- 2022].
2.Certificate of Essentiality–Intending couple/women shall apply to State Appropriate
Authority** the issuing authority of Certificate of Essentiality in Form-11 of proposed
rule, after satisfying itself, for the reasons to be recorded in writing, about the fulfilment
of the following conditions, namely-

39. a. Certificate of Medical indication in favour of either or both members of the intending couple or
intending woman necessitating gestational surrogacy from the respective District Medical Board***.
issued in Form 12 of proposed rule. [see Rule 14,the Surrogacy Rule- 2022]
b. An affidavit/order concerning the parentage and custody of the child to be born through
surrogacy, has been passed by a court of the Magistrate of the first class or above on an application
made by the intending couple or the intending woman and the surrogate mother, which shall be the
birth affidavit after the surrogate child is born. (Form 13 of proposed rule).
c. An affidavit on insurance coverage- The intending couple/women shall purchase a
general health insurance coverage in favour of surrogate mother for a period of thirty six months
from an insurance company or an agent recognized by Insurance Regulatory and Development
Authority (IRDA Act, 41 of 1999) for an amount which is sufficient enough to cover all
expenses for all complications arising out of pregnancy and also covering post-partum delivery
complication. The intending couple/women shall sign an affidavit to be sworn before a
Metropolitan Magistrate or a Judicial Magistrate of the first class giving guarantee. [Rule-5 the
Surrogacy Rule- 2022 and clause (q) of sub section (1) of section 2 of Surrogacy Act-2022]

40. 3.Certificate of eligibility- issued separately by the State Appropriate Authority,
In Form-17(intending couple ) and Form-17 A(intending woman ) of proposed rule, on fulfilment of the
following conditions-
Eligibility criteria for intending couple
(I) The intending couple are married and between the age of 23 to 50 years in case of
female and between 26 to 55 years in case of male on the day of certification;
(II) The intending couple have not had any surviving child biologically or through
adoption or through surrogacy earlier:
Provided that nothing contained in this item shall affect the intending couple who have a
child and who is mentally or physically challenged or suffers from life threatening
disorder or fatal illness with no permanent cure and approved by the appropriate
authority with due medical certificate from a District Medical Board; and (III) such other conditions as may
be specified by the regulations.

41. Steps For Surrogate Mother To Avail Surrogacy Under The Surrogacy (Regulation)
Act,2021
1.Certificate of eligibility- issued by the State Appropriate Authority, In Form-
17B (Proposed Rule) on fulfilment of the following conditions-
Eligibility criteria for surrogate mother:
(I) no woman, other than an ever married woman having a child of her own and between
the age of 25 to 35 years on the day of implantation, shall be a surrogate mother or help
in surrogacy by donating her egg or oocyte or otherwise;
(II) a willing woman shall act as a surrogate mother and be permitted to
undergo surrogacy procedures as per the provisions of this Act:
Provided that the intending couple or the intending woman shall approach the
appropriate authority with a willing woman who agrees to act as a surrogate mother;
(III) no woman shall act as a surrogate mother by providing her own gametes;

42. (IV) No woman shall act as a surrogate mother more than once in her lifetime:
Provided that the number of attempts for surrogacy procedures on the surrogate
mother shall be such as may be prescribed.
(V) a Certificate of medical and psychological fitness for surrogacy and surrogacy
procedures from a registered medical practitioner. [ Form-14 Proposed Rule]
2.Consent Form- to withdraw her consent for surrogacy before the implantation of
human embryo in her womb, [In Form-2, Surrogacy Rule-2022]
3.Consent of Surrogate for Abortion/MTP [in Form-18, proposed rule]
4.Screening of the Surrogate Mother. [in Form-19, proposed rule].

43. Dos:
They can perform any Surrogacy related Procedures.
Medical indications necessitating gestational surrogacy.- A woman may opt for surrogacy
if; -
(a) she has no uterus or missing uterus or abnormal uterus (like hypoplastic uterus or
intrauterine adhesions or thin endometrium or small uni-cornuate uterus, T-shaped
uterus) or if the uterus is surgically removed due to any medical conditions such as
gynaecological cancer;
(b) intended parent or woman who has repeatedly failed to conceive after multiple In
vitro fertilization or intracytoplasmic sperm injection attempts. (Recurrent implantation
failure);
(c) multiple pregnancy losses resulting from an unexplained medical reason. unexplained
graft rejection due to exaggerated immune response;
(d) any illness that makes it impossible for woman to carry a pregnancy to viability or
pregnancy that is life threatening.

44. Embryo
transfer act
Not more than 2 embryos are
transferred(per cycle) to minimize
multiple pregnancies
A higher number of embryos can be
transferred in 40yr age of woman
Access oocytes or embryos are
cryopreserved for further use.