Artificial / Assisted Reproductive Techniques (ART) Dr Sohani Verma Sr. Consultant Obstetrics & Gynaecology Infertility & ART Specialist Clinical & Academic Coordinator Indraprastha Apollo Hospitals, New Delhi Chairperson North Zone AICC RCOG President Elect Indian Fertility Society

Introduction A woman of reproductive age who has not conceived after 1 year of unprotected vaginal sexual intercourse, in the absence of any known cause of infertility, should be offered further clinical assessment and investigation along with her partner. Offer an earlier referral for specialist consultation to discuss the options for attempting conception, further assessment and appropriate treatment where – - the woman is aged 36 years or over - there is a known clinical cause of infertility or a history of predisposing factors for infertility NICE Guidelines 2013

Main Causes of Infertility Multiple relatively minor abnormalities, either with 1 partner or both, account for 30% of all causes

Assisted Reproductive Techniques (ART) Any treatment that deals with “means of conception other than vaginal intercourse” is termed as ART. NICE guideline 2013 IUI – Intra Uterine Insemination (Husband / Donor) IVF + ET – In Vitro Fertilization + Embryo transfer ICSI – Intra Cytoplasmic Sperm Injection

IUI Injection of washed prepared sperms into the uterine cavity through a fine catheter during peri-ovulatory phase in a natural or stimulated cycle. Although pregnancy may not occur as quickly, a policy of initial treatment by IUI will probably save 20% of couples from moving onto IVF After 3-4 cycles of failed IUI treatment, patients should be encouraged to opt for IVF

IUI The procedure may help in increasing the chances of pregnancy in following ways – Allowing sperm-ovum contact close to the date and time of ovulation By bringing the sperm very close to the site of fertilization and by passing the cervical factors Sperm preparation increases the sperm density and removes all antigens on the surface of sperm and in seminal plasma IUI is the simplest and the least expensive method of ART IUI alone (natural cycle) does not improve pregnancy chances, hence mild ovarian stimulation is usually recommended.

Indications for Intra Uterine Insemination (IUI) - At least one Fallopian tube must be normal and patent - Mild male infertility - Unexplained infertility - Ovulatory dysfunction, PCOS - Mild endometriosis - Cervical factors - Coital problems - Immunological factors - HIV, HBs Ag infection - Donor Sperm

Indications for Donor Sperm IUI Azoospermia (where ICSI is not an option) Severely subnormal semen parameters (ICSI not an option) Persistent failure of ICSI Rh Isoimmunization Hereditary disease in the male partners

Indication for ART – IUI or IVF The indications for IUI are often not dissimilar to those for IVF (or even for ICSI for moderate male factor) and often interchangeable with overlapping.

Common Indications for IUI Indications for IVF Unexplained infertility - Unexplained infertility Endometriosis (mild) - Endometriosis (moderate to severe) Male factor infertility (mild) - Male factor infertility (moderate to severe) Ovulatory disorders - Ovulatory disorders Inability to have vaginal intercourse People with conditions that require - Tubal pathology specific consideration (such as man HIV - Donor Oocyte positive) - Genetic Surrogacy - People in same-sex relationship - PGD (Possibility of genetically - Donor Sperm transmitted disease) - Fertility preservation in cancer patients - Where ICSI is indicated (Azoospermia)

Meta-Analysis of IUI in Male Factor Pregnancy Rate Timed intercourse in natural cycle 2.4% Timed intercourse in COH cycle 5.0% IUI in natural cycle 6.5% IUI in COH cycles 12.6% Cohlen BJ et al Cochrane database Syst Rev 2003

Basic requirements for IUI success Patient selection Age of female partner < 35 years Duration of infertility < 5 years Cause of infertility (at least one functional normal fallopian tube and no uterine factors) Adequate ovarian reserve (based on Serum AMH, antral follicle count, Day 2 FSH, LH, E2 levels) Semen parameters Post wash TMSC >5 million/ml Best pregnancy rates with >10 million/ml < 1 or 2 million/ml – do not waste time in IUI. Advice IVF / ICSI straight away

Basic requirements for IUI success contd… Choice of ovarian stimulation used Number of dominant follicles – 1 to 3 follicles Use of “transvaginal ultrasound follicle monitoring” Timing of IUI Between day 12 to 16 of the cycle usually highest pregnancy rates Interval from hCG injection 32-42 Hours usually recommended (range 12-60 hours) Single IUI 36 hours after hCG is usually the preferred option.

Basic requirements for IUI success contd… Semen preparation technique – Quality and expertize of lab personnel Procedure of IUI & type of catheter used Luteal support is recommended How many IUI cycles- 3-6 cycles usually recommended

INTRAUTERINE INSEMINATION – ESHRE Guidelines There is general agreement in the literature that chances of success are better after mild ovarian stimulation and the maturation of a maximum of two or three follicles. However, the cycle must be monitored by ultrasound and hormonal analysis; if there are more than three mature follicles, the attempt should be cancelled. While the concurrent use of ovarian stimulation may increase pregnancy rates, it may be at the expense of a high chance of multiple pregnancy. The majority of pregnancies occur during the first six cycles. In any case, the number of attempts should not exceed nine cycles. When assessing the duration of an IUI programme, the age of the woman must be taken into account, to ensure timely transfer to more complex treatments if indicated.

The world's first "test-tube baby", Louise Brown, has spoken of her joy at giving birth to her first child. Baby Cameron was born on 20 December’06 in Bristol, where his 28-year-old mother lives with husband Wesley Mullinder. Well over two million "test-tube" babies have been born globally since Louise's 1978 birth after IVF

IVF and ET In Vitro Fertilization (IVF) and Embryo Transfer (ET) are the basic ART for all related technology. These include: - Intra Cytoplasmic Sperm Injection (ICSI) - Assisted hatching - Pre-implantation Genetic Diagnosis (PGD) - Cryopreservation - Donor oocyte IVF programs - Donor embryo (genetic surrogacy) - Intracytoplasmic Morphologically selected Sperm Injection (IMSI) - And many more

Various steps of an IVF treatment cycle Pre IVF work-up Ovarian stimulation Monitoring Ovulation induction Preparation of sperms Oocyte retrieval In Vitro Fertilization Embryo transfer Luteal Support

IVF & ET Procedure Picture

In vitro embryo development COC at the time of retrieval M II oocyte with a PB (Mature) 2 PN embryo 4 cell embryo 8 cell embryo Fully grown blastocyst

Indication for IVF I. IVF as first line infertility treatment Tubal pathology (severe, non-repairable) Donor Oocyte Genetic Surrogacy PGD (Possibility of genetically transmitted disease) Fertility preservation in cancer patients Where ICSI is indicated (Azoospermia) II. IVF following failed cycles of IUI Usually up to six cycles of IUI with controlled ovarian stimulation are recommended, but there are situations where couples should move to IVF earlier.

Indicators for early referral I. Female age - The biological clock is the major adversary to human reproduction

Woman’s age is the initial predictor of her overall chance of success Live birth rates per Embryo transfer by age (HFEA post-October 2007 data) NICE Guideline 2013

II. Diminished Ovarian Reserve at any age - AMH- anti-Mullerian hormone of less than or equal to 5.4pmol/l Antral Follicle Count (AFC) – Less than or equal to 4 Day 2/3 FSH >8.9 IU/L Endometriosis Moderate (more than slightly abnormal) degree of semen quality abnormalities. V. Tubal Compromise NICE Guideline 2013

ICSI Unprecedented successful development of ART which has revolutionized the management of severe male infertility (Van Steirte-ghen 1992) The procedure involves the direct injection of a single sperm into the egg cytoplasm

Indications for ICSI Severe alterations of semen characteristics History of fertilization failure in conventional IVF attempts Testicular or epidydimal sperm Other relative indications

Success rates following IVF / ICSI 24.7 percent clinical pregnancies of all women who undergo IVF treatment (HFEA 2011). 50% of all embryos cultured in vitro reach blastocysts stage by day 6. About 15% of transferred embryos will develop into a baby

Basics requirements for IVF/ICSI success Pre – IVF work up of the infertile couple Clinical history Examination Investigations Counseling Why necessary? To identify the cause of infertility and thereby prognosis To identify and correct associated adverse factors before treating primary disorder To decide most appropriate treatment protocol - Type of drug - starting dosage - expected response and problems To assess reproductive ageing and plan early access / resort to ART treatments

Basic requirements for IVF/ICSI success contd… 2. To get adequate number of good quality oocytes Predictors of COHS response Normal responders Hyper responders Hypo- responders - Age, AMH, AFC - Response to earlier COHS - Basal FSH, LH, E2 - BMI, Smoking, Alcohol - Previous Ovarian Surgery B. Selection of COHS protocol - Agonist versus Antagonist protocols - Mild stimulation protocols

Basic requirements for IVF/ICSI success contd… C. Ultrasound monitoring with power and colour Doppler D. Biochemical Monitoring Ovulation induction - hCG - urinary / recombinant - GnRh agonist Technique of Oocyte retrieval Embryology lab quality and expertize IVF or ICSI Selecting best embryo (s) for transfer Number of embryos transferred Embryo transfer technique Luteal Support

Luteal Support

Luteal Support The transformation of mature follicle into corpus Luteum (CL) after the release of ovum is triggered by an optimal LH surge. The function of CL is dependent upon continued LH stimulation in luteal phase. CL is an essential source of pro-fertility hormones ie Progesterone (P), Estrogen (E) and other vasoactive and growth factors.

Luteal Support It is well established that the ovarian stimulation regimens used in assisted reproduction cycles alter the luteal phase. Edwards et al 1980, Kolibianakis et al 2003 Ovarian stimulation causes an inadequate development of the endometrium an asynchrony between the endometrium and the transferred embryo and adverse effects on endometrial receptivity Macklon & Fraser 2000, Devroey et al 2004

Luteal Support contd… The luteal phase defect in IVF is present whether GnRH agonist or antagonist is used (Friedlers et al 2006). The possible mechanism responsible may be – Continuation of pituitary down regulation effect Duration of luteal phase is shortened Formation of multiple CL leading to inhibition of pulsatile LH release Loss of granulosa cells during oocyte retrieval

Luteal Phase Support Endometrial support – complements production by CL Progesterone preparation Estrogen preparation Agents which support CL hCG GnRH-analogue LH Newer agents which promote angiogenesis and vascular supply

Progesterone preparations available Micronized Oral / vaginal - 200-400 mg BD Vaginal Gel (8%) - 90 mg daily Vaginal Pessary - 100-400 mg daily Intramuscular (oil based) - 100-400 mg daily (iii) Subcutaneous (aqueous preparation) - 25 mg daily (iv) Synthetic – Dydrogesterone - 10 mg BD or TDS

Estrogen as an adjuvant to LPS Estradiol valerate. Hemihydrate Oral (intravaginal) 2-6 mg/day Micronized Estradiol Oral or intravaginal Transdermal Estradiol Patches (2 per week) 50-100 ugm/day

GnRH agonist as an adjuvent to LPS Luteal Phase Support for assisted reproduction cycles (Cochrane Review 2011) Tesarik J et al 2006 published their result on 600 women randomly assigned to receive a single injection of GnRH agonist (0.1 mg of triptorelin) or placebo on Day 6 after ICSI. The results showed improvement of implantation and live birth rates. Van der Linder et al investigated progesterone versus prog + GnRHagonist Six studies (1646 women) There were significant results showing a benefit from addition to GnRH agonist to progesterone for the outcomes of live birth, clinical pregnancy and ongoing pregnancy.

Luteal Phase Support for ART Cycles Authors' conclusions Cochrane review 2011 showed a significant effect in favour of progesterone for luteal phase support, favouring synthetic progesterone over micronized progesterone. Overall, the addition of other substances such as estrogen or hCG did not seem to improve outcomes. They found no evidence favouring a specific route or duration of administration of progesterone. It was found that hCG, or hCG plus progesterone, was associated with a higher risk of OHSS. The use of hCG should therefore be avoided. There were significant results showing a benefit from addition of GnRH agonist to progesterone for the outcomes of live birth, clinical pregnancy and on-going pregnancy. For now, progesterone seems to be the best option as luteal phase support, with better pregnancy results when synthetic progesterone is used. Cochrane Review 2011

Nutritional Supplements and ART outcome No definite conclusive evidence Anti-oxidants – Vit C, E, selenium, zinc, taurine, carotene, lycopene Vitamins – Folate, Vit B 12 Myoinositol and D-chiro-inositol (vit B complex) L – Arginine DHEA

Dehydroepiandrosterone (DHEA) supplementation Cason and associates (2000) were first to suggest therapeutic benefits from the supplementation of DHEA in women with diminished ovarian reserve and suggested it may improve oocyte yields via IGF-1. It was left to a 43 year old infertility patient in US (advised donor oocytes) to discover their paper and self administer DHEA while undergoing subsequent IVF cycles. The patient underwent nine consecutive IVF cycles and increased oocytes and embryo yields from cycle to cycle, starting with one egg and embryo, respectively, and ending up with 17 oocytes and 16 embryos in her ninth cycle. (Gleicher et al 2009)

Dehydroepiandrosterone (DHEA) supplementation While all other pharmacological agents affect follicle maturation only during the final stage – gonadotropin – sensitive last 2 weeks, DHEA in contrast appears to affect folliculogenesis at much earlier stages of in-vivo follicle maturation (Gleicher N etal 2011) DHEA has been shown to increase the number of primary, preantral and antral follicles. DHEA supplementation is reported to improve ovarian response, IVF parameters and pregnancy chances. Younger patients with POA appears to have a slight pregnancy advantage.

Cumulative pregnancy rates in women with DOR with and without DHEA supplementation. POA patients appear to have a slight pregnancy advantage, Barad et al 2007

DHEA supplementation is also shown to significantly (50-80%) reduce the miscarriage risks in patients with poor ovarian reserve (Gleicher etal 2007) Age-stratified miscarriage rates in DHEA supplemented DOR patient in comparison to national U.S. IVF pregnancies. Gleicher et al 2009

Treatment protocols, side effects and complications Micronized DHEA at a dosage of 25mg TID Effects occur relatively quickly (6-8 weeks) but peak only after 5-6 months of supplementation. Side effects are small and rare and primarily relate to androgen effects – oily skin, acne vulgaris and hair loss. Even long-term therapy of DHEA in suggested dosages have been demonstrated safe (Panjari M etal 2009). However, before declaring DHEA as a wonder drug, larger RCTs are urgently needed to confirm the benefits.

Thank You Sohani Verma