Indi an Journal of Experimental Biology Vol. 39, January 2001, pp. 1-10

Review Article

Potential researchable areas in ARTs--Oocyte maturation and embryo development

Tarala D Nandedkar & Radhika L Kelkar

Institute for Research in Reproduction, Mumbai 400 012, India.

In fertility is a co mmonl y encountered situation occurri ng equally in both sexes. In vitro fertilization and embryo trans­fer (IVF-ET) and other ass isted reproductive technologies (A RTs) have enhanced the possibilities for successful treatment to tackl e in fert ility. However, ARTs currently face limitations due to the fact that although success rate is high for the initial stages such as ovulation induction and fertilization , it dwindles progressively so that the success rate of a take home baby is as low as I 5-20%. Research centred around various stages in an IVF programme is therefore necessary to devise protocols that ensure a hi gher success rate. This review takes a look at the potential areas currently under research in the field of ARTs, such as , in vitro oocyte maturation, oocyte/embryo cryopreservation , embryo culture, preimplantation genetic diag­nosis. Their applications, in clinical conditions such as cancer, have been di scussed.

Infertility is a problem that affects men and women , the causes and prevalence rate of which may vary. The desire to reproduce is a highly motivating force and failure to do so could prove to be a catastrophic experience for the couple. Defined as a disease or condition diagnosed after a couple has had 2 years of unprotected coitus without conception; infertility is quite common with one in every five couples being affected at some point of time. While approximately 40% cases are due to the male factor and 40% due to female factor, in the remainder 20% the causes may be unexplained .

Under normal conditions, the course of events that culminate into a pregnancy include-ovulation, fertilization , cleavage and implantation (Fig. 1). During each menstrual cycle, an oocyte from a dominant follicle (selected from a pool of developing follicles) is released into the fallopian tube. Following ovulation , fertilization occurs as a result of a single sperm cell penetrating the barriers surrounding the oocyte, thereby allowing fusion of male and female pronuclei. The zygote so formed is transported from the tube to the uterus during which it undergoes multiple and rapid cleavages to form a mass of cells called morula. In the uterus , the morula undergoes further divisions to form the blastocyst, so that, by day five or six post-fertilization, the trophoblast cells invade the uterine wall and implant the embryo for further growth. Complex molecular interactions directed by hormonal signals underlie the seemingly simple processes mentioned above and any disturbances in the same could be a cause of infertility

in the female. Thus, female infertility may broadly be due to-{i) Blockage of fallopian tubes preventing fertilization; (ii) Absence of ovulation; (iii) Hormonal deficiency ; and (iv) Non-receptive uterine lining.

Assisted reproductive technologies (ARTs) such as in vitro fertilization and embryo transfer (IVF-ET), gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFf), intra-cytoplasmic sperm injection (ICSI) can help the infertile women to overcome the barriers to fertility. However, ARTs do face certain limitations with age and ovarian responsiveness, at play! . While fertilization rate is as high as 80-90%, the success rate of a take-home baby is 15-20%. It is therefore necessary to conduct research in areas such as oocyte maturation, embryo development and implantation to develop protocols that ensure a higher success rate.

Fig. !- Diagrammatic representation of events during early preg­nancy following ovulation : Release of the egg from the ovary; fertili zation and cleavage in the fallopian tube and transfer of the embryo to the uterus and implantation .

2 INDIAN J EXP BIOL, JANUARY 2001

Oocyte maturation Oocyte retrieval and culture-The first successful

human pregnancy from IVF-ET is to make use of a single oocyte retrieved during a natural (non­stimulated) cycle. The time of aspiration is decided by monitoring LH levels every 3 hours2

• However, the low success rate associated with this approach led to the research in 1980s that resulted in the development of a number of stimulation treatment protocols. Use of GnRH agonists to down-regulate the pituitary followed by ovarian hyperstimulation using human menopausal gonadotrophin (HMG) and superovulation by HCG is a commonly used protocol in IVF programmes. However, these stimulation protoco ls have a number of disadvantages such as high cost of hormones, multiple side-effects and a risk of ovarian hyperstimulation (especially in case of Polycystic Ovarian Syndrome patients).

In view of the above, development of alternati ve treatment modalities for obtaining oocytes for IVF has been a subject of intensive research in the past decades. Two important strategies that have been developed and being further investigated include-{a) In vitro oocyte maturation (IVM) i.e. retrieval of immature oocytes (1 00-120 J..lm diameter) from antral follicles for maturation in vitro; and (b)ln vitro follicle growth (IVG) i.e. growth of primordial or preantral follicles to full size for subsequent IVM.

Immature human oocytes and intact follicles have been obtained from ovarian tissue removed surgically and cultured in vitro3• Trounson et al. developed a reliable in vivo method of transvaginal ultra-sound­guided oocyte retrieval\ which has been further refined by Russell et aZS. The requirements of medium, nutritional factors, macromolecular and hormonal supplements and culture conditions for oocyte maturation have been reported by several workers and presented in a number of recent reviews6-8.

The first viable pregnancy using IVM oocytes was reported by Cha et al. in 1991 followed by subsequent reports45

·9

•10

• Similarly, attempts to grow primordial follicles to Graafian stage have met with some success as shown by studies of Eppig and O'Brien in mice 11

, and Hovatta et al. 12'13 and Oktay etal. in

humans 14• However, a major problem that needs to be

tackled is the reduced developmental competence of oocytes matured in vitro.

The process of oocyte maturation involves two close ly integrated yet independent processes i.e. nuclear maturation and cytoplasmic maturation.

nuclear maturation involves retntttatiOn of meiosis, germinal vesicle breakdown (GVBD) and meiotic progression from metaphase I to metaphase II. The process of cytoplasmic maturation involves changes, such as, relocation of cytoplasmic organelles and alterations in membrane transport systems that prepare the oocyte for subsequent fet1ilization and embryogenesis. Any imperfections in these processes of oocyte maturation could subsequently compromise embryo viability.

An essential pre-requisite to oocyte maturation is the synthesis and storage of a vide range of molecules, both in the oocyte and cumulus cell s, during oocyte growth. The mRNA and proteins accumulated in the growth stage are necessary to activate the maturation promoting factor which in turn drives nuclear maturation 15

•

The molecular reprogramming that leads to oocyte maturation is under the regulatory control of signals produced by cumulus cells, the latter being produced in response to gonadotrophin stimulation. Moor et al. 16 thus suggest that the key to oocyte maturation and embryo viability in vitro resides in the follicular compartment rather than the oocyte. While Ca2+,

inositol-! , 4, 5-triphosphate, cyclic AMP, are known to mediate cross-talk between oocyte and cumulus cells during GVBD, factors that fine-tune meiotic progression from metaphase I to metaphase II are unclear. A number of candidate factors have been suggested to play a role in follicular signalling that directs oocyte maturation, such as, oestradiol 17B, EGF, activin, stem cell factor, thyroid hormone, Mos protein (product of c-mos) matrix metalloproteinases (Fig. 2) and have been extensively reviewed 15- 17 •

Oocyte diameter at the time of collection is another factor that determines meiotic potential of oocytes in vitro, as shown by Durinzi et al18.

Follicular fluid steroids and proteins - markers of oocyte quality--Steroid hormones present in the ovarian follicular fluid are believed to play an important role in oocyte maturation and acquisition of developmental competence of the embryo 16

•

Consequently, estradiol levels are often monitored for detecting matured follicles in gonadotrophin­stimulated ovulation protocols. Besides being used as a marker for ovulation induction, local estradiol levels in the follicular fluid may also be used as a predictor of pregnancy outcome 19

• Progesterone, an indicator of ovulation and luteal function, can also be of a prognostic value in pregnancy by IVP 0. In a recent study, a correlation between human chorionic

NANDEDKAR et al. : POTENTIAL RESEARCHABLE AREAS IN ARTS 3

gonadotrophin (HCG) in follicular fluid and granulosa cells to oocyte maturity and fertilization has been reported2 1

.

In addition to sex steroids, proteins and peptides in the follicular fluid can help in determining the quality of oocyte retrieval for ARTs and success of pregnancy. The role of inhibins and activins in human ovulation, conception and pregnancy have been extensively studied and reviewed22 . Many recent studies have reported the potential use of these two peptides as markers of ovarian reserve and oocyte quality23-25•

Oocyte preservation--During aspiration of oocytes from ovaries of patients with stimulated cycles, as many as 10 oocytes can be procured at a time. All these may not be used for IVF and could be preserved for revival and use in the next cycle.

Cryopreservation is a slow freezing method used for preserving embryos/oocytes using various cryoprotective agents (CPAs) such as ethylene glycol, dimethyl sulphoxide (DMSO), 1, 2 - propanediol (with or without sucrose) etc . These agents depress the freezing point thereby reducing the extent of intracellular ice formation and minimising the extracellular salt concentration at a given temperature26

.

Despite the efforts of numerous workers, human oocyte cryopreservation still remains a research protocol with a success rate as low as 1% of living babies per thawed oocyte27

. A number of reasons could be attributed to the same, such as, low fertilization due to a weakened zona, disorganisation of the spindle apparatus and high incidence of polyploidy15

•28

·29

. While zona drilling and ICSI are

--1-\-- >--Stem cell factor

Activin

being successfully used to overcome problems related to the zona, solutions to prevent spindle disorganisation, polyploidy etc. still remain elusive.

The process of vitrification may add a new dimension to oocyte cryopreservation. While freezing involves precipitation of water from the solution; vitrification implies transition of an aqueous solution as a whole from liquid to glass state (solid) bypassing crystalline state26

• In a recent study by Isachenko and Nayudu, the effect of temperature and egg yolk on chromatin and spindle normality and cumulus integrity was assessed, in mouse GV oocytes. Results of the study ' have indicated that both pre- and post­freeze exposure of GV oocytes to 37°C with inclusion of egg yolk has a positive effect on oocyte and cumulus cell integrity with >80% normal survival post thaw, 84% maturation rate and 45% normal spindle configuration30•

New approaches such as above may offer solutions to some problems currently shadowing the field of cryopreservation and in combination with IVM may pave the way for the establishment of oocyte banks facilitating oocyte donation for clinical cases of premature menopause such as Turner's Syndrome31 .

Another application of cryopreservation is in case of cancer patients (Fig. 3). Aggressive chemotherapy and/or radiotherapy for malignancies may compromise ovarian function by depleting follicle store thereby leading to infertility. In such cases, cryopreservation of ovarian cortical tissue is one strategy that holds promise for these patients in order to restore fertility. A number of approaches for the use of cryopreserved tissue are currently being standardised since a substantial number of primordial

LH

-1-

"" Activin

Fig. 2--Possible factors regulating oocyte maturation.

4 INDIAN J EXP BIOL, JANUARY 2001

follicles are found to be morphologically normal and viable after freeze-thawing. Autografting the tissue at an orthotopic site (laparoscopic insertion of cortical biopsies into surgically resected ovary) or heterotopic site with a rich vascular bed such as the kidney are some of the options. These, however, may pose a risk of reintroducing the malignant cells to the patient in remission, especially in case of blood-borne or ovarian cancers. Follicle isolation from thawed tissue for in vitro growth and maturation is a possible alternati ve, however, much remains to be standardised in this approach29· 32 .

Embryo development

Embryo culture and transfer---ln human IVF-ET programmes, intra-uterine embryo transfer is routinely carried out on day 2 or 3 of development, with an implantation rate of t5%. An obvious alternative for increas ing implantation rate would, therefore, be the extended culture and transfer at blastocyst stage. This will not only help in the identification of those embryos with little or no developmental potential , but will also facilitate synchronization of the embryo with the female tract. Further, since the blastocysts have a higher implantation rate than cleavage stage embryos, fewer embryos can be used for transfer, reducing the risk of multiple gestations which is a major problem currently faced by IVF clinics24

•

Inspite of these proposed advantages, success rate even in case of blastocyst transfer is lower, one plausible reason being the failure to mimic adequately in culture, the environment that the embryo encounters in vivo. During its passage through the oviduct and uterus, the embryo is exposed to dynamic changes in the environment. Two approaches are often resorted to for mimicking the same i.e. (i) sequential changes in culture media, (ii) co-cultures with epithelial cells.

Culture in sequential media--The concept of sequential media is based on the dynamics of embryo physiology and the everchanging requirements of the embryo for two key nutrients. i.e. carbohydrates and amino acids, wherein lies the need for the use of more than a single culture medium. For instance, Gardener and Lane have reported the use of two culture media namely Growth 1 and Growth 2 (G 1 and G2) for human embryo culture, up to blastocyst stage. While G 1 medium is based on the carbohydrates and amino acids present in the human fallopian tube at the time of cleavage, G2 is based on conditions in the human

uterus during blastocyst development and differentiation33

.

An important component of all sequential media is glycine which is catabolized into glycolate and glyoxylate with release of ammonia. The latter, in vitro, may form its carbonate or bicarbonate that are highly unstable in alkaline pH. Glutamine is another component of the medium and is needed before genomic activation. Initially, glucose was used as the main carbohydrate source in all media. Recently a trend has emerged for reduction or complete omission of the same from the culture medium wi th pyruvate as the subsequent replacement34

. However workers differ in thei r opinion regarding the importance of glucose in the medium35 . Pyruvate has been advocated not only to act as an energy source but also to detoxify the ammonia in the embryo by transamination to form alanine.

Equally important is the inclusion of albumin, ethylene diamine tetraacetic acid (EDTA), insulin, epidermal growth factor (EGF) in the medium. While EDTA is added in the first step as the free radical scavenger, it is omitted in the second step of culture due to its effect on genomic activation. On the other hand, growth factors are generally added in the second culture medium36•

Co-cultures-Co-culture, as the term implies, is the culture of the developing pre-embryo with epithelial cells with a view to facilitate potential dynamic interactions between the two. The cell . types commonly used include tubal or endometrial

Grafting

~ Ovarian ti ssue slices ~ (Cortical biopsy)

Cryopreservation

Thawing

Ma;.nual I Enzymatic isolation of follicles

Autogra ft Xenograft l Orthotopic

site

Natural fertility

l -01 SCID Preantral follicles

Transplant 1 ~ at hete.rotopic ~

stte ~

L .Q-· Grow~ muturation IVF-ET ~(--- of oocyte in -vitro

Fig. 3--Use of cryopreserved ovarian tissue to restore fertility in cancer patients.

ANDEDKAR eta/. : POTENTIAL RESEARCHABLE AREAS IN ARTS s

epithelium (of human or animal origin) autologous human cumulus or granulosa cells or African green monkey kidney cells (vera cell line). A number of studies evaluating the effect s of these cell types on the developing embryo report an increase in the average number of blastomeres, decreased fragmentation and hence improved embryo development37

·38

. In one such study, a significant improvement in the average number of blastomeres per pre-embryo has been reported, using an endometrial co-culture system set up using one passage, freeze-thawed stromal and glandular cells obta ined from the endometrial biopsy of the patient. However, the implantation and pregnancy rate has been found to be the same between the co-culture and non-co-culture cycles in patients with repeated IVF failures38

.

In contrast to thi s, some groups report no difference in mea n cell number per pre-embryo and blastulation rates using bovine oviductal or vero co­culture systems39 .. 0.Yet another effect of co-culture seems to be improvement in the hatching rate and implantation rate, where the former effect can be attributed to the thinning of zona pellucida observed during co-culture4 1

• On similar lines, co-cultures in combination with assisted hatching seem to produce beneficial effects in patients with poor prognos is or repeated IVF failures , suggest ing that this approach may hold a potential solution to the problem of implantation failure due to impaired hatching42

·43

.

An important issue to be taken into consideration is the risk of bacterial or viral infection through the use of human heterologous cells or animal cells recovered at the slaughterhouses or established cell lines. Use of synthetic medi a that give a co-culture e ffect has been suggested as an alternative in thi s case4 1

•

Factors regulating embryo development

Following fertilization , the egg undergoes a series of cleavage di visions to form the blastocyst. However, a significant number of embryos die during thi s preimplantation period for reasons unexplained. Several possibilities for the occurrence of the same have been explored, includi ng chromosomal abnormalities . genetic defects , unfavourable uterine env ironment etc.

Aneuploidy, haploidy, polyplo idy, mosaicism are some of the numerical chromosomal abnormalities present in deve loping manJinalian e mbryos44

• A study by Viuff et a/. of bovine blastocysts developed in vitro reports a high proportion of mixoploidy in these blastocysts45

• The significance of the phenomenon of

chromosomal abnormalities in prei mplantation human embryos is reflected in a study wherein approximately half of the spontaneous abortions in the first and second trimester are chromosomally abnormal46

•

During IVF, embryo quality is determined by two main criteria name ly, the rate of development and degree of fragmentation. The early stages of post -fertilization development are controlled by maternal factors stored in the egg. At some point during development, the genetic information source is switched to zygotic genes. Thi s switching of genetic source or zygotic gene activation (ZGA) is reflected in the alterations in protein expression pattern in mouse oocytes and preimplantation embryos47 The mouse Ped gene is a widely studied gene 111

preimplantation embryos. Localized to Q region of the major histocompatibility complex (MHC). it

Antral follicle

Oocyte retrieval.

,!, ~Polar bo: biopsy

IVF-ET

,!, 16- 18h

@ 2-PN Oocyte

(Zygote)

In-vitro culture

PGD

Day 3 ~ Post·insemm~tion

lffl'-x----+ Blastom<re biops~ 1 - ~ blastomorcs)

V;£0 PGO

6-10 cell stage

Day 5-6 ~ Post-insemination

C\--+Biastocyst biopsy .. (upto 10 troplectoderm cells)

Blastocyst

Fig. 4---Methods to procure genetic material for PGD.

6 INDI AN J EX P BIOL. JANUA RY 200 1

encodes the prote in Qa - 2 antigen that determines the rate of cleavage in the embryo. The e mbryonic Ped gene is seen to be acti vated only after 2 - cell stage, which is the stage of ZGA in mouse48

• The search for a human homologue of the mouse Ped gene, however is not complete.

A very common phenomenon observed during pre impl antation development is the complete or parti al fragmentation of one or more blastome res. T here is a general consensus that degree of fragmentation and developmenta l competence are in versely related. Both necros is and apoptos is have been suggested as possible mec ha nisms resul ting in fragme ntat ion. W hile the fo rmer is a resul t of a physical or chemica l inj ury, the latter is genet ica ll y regulated. Conseque ntly, stud ies have foc ussed on two maj or gene fam ilies involved in apoptos is , namely the Caspase and Bel - 2 fa mil y. Studies to assess mRNA leve ls for apoptotic genes report caspase mRNA in oocytes only while caspase proteolytic activity in polar bodi es and fragme nted zygotes49 On the other hand, bax mRNA has been found in all stages (incl ud ing oocyte) un like Bcl-2 mRNA wh ich has been detected onl y in zygote and subsequent preimplantation stages48

. Interesti ng ly, Bcl-2 protein levels are lower in fragmented blastocysts as compared to norma l ones suggesting a role for Bc l-2 and bax in apoptotic embryos49

.

However, in a study by Antczak and B lerkom, no correlation between apoptosis and fragmentati on can be establ ished. Instead an assoc iat ion has been fo und between patte rn s of fragmentation and the parti al or near-tota l loss of certain regulatory protei ns that occ ur in polari zed domai ns in the mature oocyte and embryo as we!J5°.

Assisted hatching-The transfer of' the embryo to the uterus is fo ll owed by impl antati on, whereby the blastocys t attaches itself to the uterine wall for further development. An essential pre-requi s ite to implantation is the hatching of the blastocyst through a series of contrac ti ons and expansions of the blastocyst and rupture of zona pe llucida. Consequently , impa irment of hatc hing has been suggested as one of the reasons fo r a low impl antation rate. Ass isted hatch ing is often resorted to in order to overcome the same. A number of procedures are employed for th is purpose , such as partia l zona dissect ion (PZD) i.e. creating breaches in zona mechanically; zona drill ing usi ng acid Tyrode's solution ; or employi ng a laser beam with di rect contact (UV) or without direct contact (IR). T hese

techniques have the ir own share of advantages and di sadvantages, however, they have been reported to improve implantation rates51.

Pre-implantation genetic diagnosis Pre-implantation genetic diagnosis (PGD) as the

term suggests, is the genetic analys is pri or to impl antati on carri ed out to screen for e mbryos with seri ous genetic di seases52 • It is an earl y form of prenata l diagnosis which he lps in the se lect ion of unaffec ted embryos for impl antati on o r at times, for sex-based selection of the embryo for transfer. PGD offers the high-ri sk couples the option of decreasing the ri sk of transmitting inhe ritab le genet ic d iseases to the offspring.

It has been nea rl y a decade since the first pregnancies were es tab lished after emp loying PGD to screen for an X-linked defec t in couples at risk5

'.

S ince then PGD has been used successfull y to screen fo r s ing le gene defects of chromosomal abberrations . In case of sing le gene defects with known gene­struct ure, such as cystic fibros is o r Tay Sachs. D A obtained from the e mbryo is analysed fo r the presence of defect ive genes. On the other hand, if the gene defec t is not known , a~ in case of Ducc hene M uscular Dystrophy (DMD) or he mophili a. the sex of the embryo is determined (s ince these defects occur in ma les) and onl y fema le e mbryos are transferred. C hromosomal abnormaliti es such as Down 's Synd rome are screened by ka ryotyping i.e . cytogenetic analysis of banded metaphase chromosomes .

Sources of DNA for analysis- T he DNA sample needed fo r PGD may be obta ined (Fig. 4) e ither by biopsy of (i) first po lar body from the unfe rtili zed oocyte, or (ii ) one or more blastomeres from c leavage stage e mbryo, or (iii ) trophectoderm cell s from the bl astocys t.

( i) Po lar body bi opsy-The fi rst po lar body formed as a res ult o f the first me iotic di vision of the oocyte can be analysed within 24 hr after the oocyte is re trieved. Thi s approach is often more ethicall y acceptable since the embryo is left untouched. However, the analys is in terms of the e mbryo is indirec t since the polar body analysed will contain the genotype of the oocyte on ly. Consequently, the method faces a drawback in that autosomal dominant d isease in the male or sex determination cannot be carried out.

(i i) B lastomere biopsy-Here, one or two blastomeres are removed from the embryo at the 6-8

NANDEDKAR et a/. : POTENTIAL RESEARCHABLE AREAS IN ARTS 7

cell stage (or day 3 post-fe rtilization) and analysed . Subsequently, embryos determined to be unaffected are transferred to uterus at the end of day 3.

This is the most preferred approach since most embryos attain this stage in vitro and since blastomeres are totipotent, removal of one or two blastomeres does not affect further embryo development. This approach, however, falls short in that only one or two blastomeres can be used, which at times may not be sufficient 111 cases of chromosomal mosaici sm44

.

(iii) Blastocyst biopsy-Compared to the above two approaches, blastocyst biopsy offers much more material for analysis, wherein upto ten trophectoderm cells can be removed from the embryos cultured upto day 5 or 6 post- fertilization. However, this approach too has its shortcomings in that very few embryos grow upto this stage in vitro and there is a possibility that the biopsied trophectoderm cells may have diverged genetically from the inner cell mass which will later form the fetus.

Analytical techniques in PGD-The tools for PGD may be divided into two categories based on direct analysis of DNA, such as karyotyping, polymerase chain reaction (PCR) and in situ hybridisation (ISH), or an indirect analysis through gene products54. The latter for instance may involve biochemical assays to monitor enzyme activity during various developmental stages. However, no assays have been standardised so far in human, probably due to the activation of embryonic genome taking place relatively late in development.

Karyotyping involves cytogenetic analysis of metaphase chromosomes for chromosomal aberrations. The technique has its limitations due to the time consumed and difficulty in obtaining cells in metaphase. In this context, a recent study by Willasden et al. may provide a rapid method for karyotyping. The present study involved fusion of human blastomeres with in vitro matured bovine oocytes with a view to force the blastomere nuclei into metaphase using the metaphase inducing factors present in unfertilized eggs. Of the 66 successfully fused hybrid cells with human chromatin, 64(97%) yielded chromosomes that could be karyotyped . Amongst the embryos from two patients with maternal translocations, one embryo was found normal and hence transferred leading to one pregnancy with normal fetus. Studies such as these may provide a basis for a rapid method for

karyotyping blastomeres from preimplantation embryos55 .

The polymerase chain reaction (PCR) is usually used for detecting single gene defects . Most PCR -based techniques make use of nested PCR, wherein two rounds of PCR are carried out and the oligonucleotide primers used in the second round are specific for sequences internal to those sequences recognised in the first round . This technique offers several advantages such as increased sensitivity and specificity and a reduced risk of carry-over contamination. Fluorescent PCR, a modification of PCR involving fluorescent nucleotide analogues incorporated into the primers or amplified products, helps in increasing sensitivity and accuracy of detection . PCR - based techniques are be ing used effectively for the detection of a number of single gene defects causing diseases such as cystic fibrosi s, Ducchene muscular dystrophy, Tay Sachs disease, sickle-cell anaemia, B-thalassaemia, Lesch-Nyhan syndrome, Huntington's disease etc.

Fluorescence in situ hybridisation ([F] ISH) is the most reliable technique for detecting chromosomal abnormalities , both numerical and structural , in pre­implantation embryos, using fluorescent DNA probes. The procedure involves spreading the single embryonic nucleus on a slide with subsequent ISH using fluorescent chromosome specific probes that produce different colours for each chromosome analysed. It is used widely for sex determination since it can detect both X and Y chromosomes within a single cell. Multicolour FISH (m-FISH) is a modification of this technique using which, upto five different chromosomes can be detected in a single cell. Another modification known as primed in situ labelling (PRINS) employs chromosome-specific oligonucleotide primers for sex determination in case of X-linked diseases.

Although applications of PGD are manifold in ARTs, it raises a number of ethical, legal and moral dilemmas52. Despite these, there is a general consensus among workers in this field that PGD is the need of the day.

Embryo cryopreservation During stimulation protocols, a considerable

number of mature oocytes can .be retrieved. Consequently following subsequent fertilization and embryo development, most IVF programmes resort to embryo cryopreservation . The technique offers the patients a solution for reducing the risk of multiple

8 INDIAN J EXP BIOL, JANUARY 2001

pregnancies without destroying excess embryos which can be used for subseque nt transfers, if needed, thereby reducing the need of expensive and time consuming gonadotrophic stimulation. The procedures and cryoprotective agents employed are simil ar to those mentioned previously for oocyte preservati on, however, the tech nique rende rs sati sfac tory results in case of e mbryo cryopreservati on even today, as compared to oocyte freezing20

. Kaufman et al. have reported a promi sing clinica l pregnancy rate of 21.7% in a study in volving 1239 thawed blastocysts56

. However, negative reports do ex ist, the reasons for which could possibly be attributed to two main factors - the process of cryopreservation and CPA empl oyed. Two contrasting stud ies can be cited in thi s context. Park et al have de monstrated hi ghe r e mbryo surviva l rates in expanded and ea rl y hatching bovine blastocysts by vitrification using e lectron microscope grids as contai ners57 On the other hand , a comparison of the effects of two cryopreservation procedures name ly, conventi onal slow controlled rate freezing and vi tri fica ti on was carri ed out by Uec hi eta!. Thi s study has reported a reduced functi ona l viabi lity in terms of cr-I) 2-deoxyglucose uptake and bl astocyst impl antati on rate in the vitrified group5

R.

With the increasing use of PGD, an important iss ue to be considered is whether the deve lopme ntal potential of embryos is impa ired afte r manipulati on proced ures such as drilling or biopsy and cryopreservation. So far, no human pregnancy has been reported usi ng biopsied , cryopreserved, thawed e mbryos and limited data on thi s aspect is available . In a recent study by Jo ri s et al. , the effects of e mbryo biopsy procedure on survi va l after cryopreservation has been assessed59• A significantly lower number of bl astomeres is intact in the biopsied g roup as compared to the control group; and the survival rates of biops ied e mbryos are red uced as well. However, the surviving e mbryos developed to the blastocyst stage ill vitro suggesting a possibility of their developme nt to term by dev ising better cyropreservation protocols .

Although widel y e mployed, human e mbryo cryopreservation raises many legal and ethical issues. For insta nce, the creation of orphan embryos and thei r disposal raises moral and eth ical dile mmas . In view of these, oocyte cryopreservation seems to be a preferable ::dternati ve.

Despite technological advances in ARTs, invest igators in th is field face two maj or problems.

These are--{i) the increasing numbers of cryopreserved embryos, and (ii) multiple gestati on rates. Although legislative actions have been taken in some countries to tackle them, these problems demand clinical and sc ientific soluti ons. The incidence of multiple implantation brings with it a physiological need for fetal reduction and an increased risk of loss of pregnancy. For minimi si ng this ri sk, it has been suggested that the number of embryos transferred should be restric ted to one or two and the re mainder cryopreserved. Thi s however, would in turn contribute to the problem of cryopreserved embryos. The need of the day, is therefore, to explore other alternati ves by conducting research in areas such as ill vitro oocyte maturati on and oocyte freezing. The story of ARTs would be a true success story , if researchers would develop protoco ls for successful s ing le e mbryo transfer, e nabling IVF to trave l a full circle back to the birth of Louise Brown. The first human IVF baby born as a result of a s ing le e mbryo transfer.

Acknowledgement

The authors acknowledge the encouragement and suggestions by Dr H S Junej a, Director, IRR. Mumba i, India.

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7 Salha 0 , Abusheika N & Sharma V. Dynamics of human fol ­licular growth and in vitro oocyte maturation. Hu111an Reprod Update, 4 ( 1998) 816.

8 Smitz J & Cortvrindt R, Oocyte in vitro malllration and folli ­cle culture : Current dinical achievements and future direc­tions, Jlulllan Reprod, 14 (Su ppl I) (1999 ) 145 .

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12 Hovatta 0 . Silye R. Abir R, Krausz T, Hardy K & Winston R M L, Extracellul ar matrix improves survi val of both stored and fresh human primordial and primary ovarian follicl es in long term culture, Human Rep rod, 12 ( 1997) I 032.

13 Hovatta 0. Wri ght C. Krausz T, Hardy K & Winston R M L, Human primordial , primary and secondary ovarian follicles in long - tet m cu lture : Effect of partial isolation , !Iuman Re­prod. 14 (1999) 2519.

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15 Cha K Y & Chian R C. Maturation in vitro of immature hu­man oocytcs fo r clinical use. /Iuman Reprod Update, 4 ( 1998) 103.

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22 Lockwood G M, Muttukrishna S & Ledger W L, lnhibins and activi ns in human ovul ation , conception and pregnancy. 1/u­nwJI Rep rod Update, 4 ( 1998) 284.

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24 Hall J E. Welt C K & Cramer D W. lnhibin A and inhibin B reflect ovarian func tion in assi sted reproduction but arc less useful at predicting outcome. Huma11. Reprod, 14 (1999) 409.

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26 Kritscr J K. Agca Y & Gunascna K T. The cryobiology of mammal ian oocytcs. in Reproducti i'C Tissue Banking - Scien­riJic Principles . edited by Karow A M. Critser J K (Academic Press, London) 1997, 345.

27 Mandelbaum J. Bela"isch - All art J, Junca A. Antoine J, Plachot M. Alvarez S, AI not M. & Salat- Baroux J. Cryoprc-

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29 Newton H. The cryopreservation of ovarian tissue as a strat­egy for preserving the fertility of cancer pati ents. !-Iuman Re­prod Update , 4 ( 1998) 237.

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33 Gardner D K & Lane M, Culture of viable human blastocysts in defined sequential serum - free medi a. !-Iuman Reprod, 13 (Suppl3) ( 1998) 148.

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35 Bavister B D, Glucose and culture of human embryos, Ferri/ Steril, 72 ( 1999) 233.

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45 Viuff D. Rickards L, Offenberg H. Hyttel P. Avery B. Gre\'e T, Olsaker I, Williams J L, Callcsen H & Thomsen P D. A

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