IVF & Genetic Center Jabalpur Madhya Pradesh, India

Dear Colleagues,

Hello & Best wishes for forthcoming Holi.

In this issue we are discussing monitoring the induction protocol . Any sort of handling the menstrual cycles disturbs the normal balance of endocrine. To get a success in IUI and IVF we try to make more number of eggs in woman who is already producing one egg every month naturally. We use various inducing drugs for that and try to create an environment so that the amount of FSH in the early pre-ovulatory phase increases. More FSH in this time of cycle will propel more recruiting eggs and thus we get a pool of 17-18 mm follicles . When they are triggered by surrogate LH surge ( by hCG injection ) ,chances of fertilization increases and so the chances of pregnancy.

But how to use these drugs and how to monitor that menstrual cycle is very important. There should be better case selection for using these drugs ,so that they can be justified .

Things become more confusing when we treat a dysovulatory woman like that of Poly cystic ovarian disease.
The topic is influenced by a paper from Tawfik, A. etal, Deptt. of Ob gy, university cantonal hospital,Geneva, Switzerland. Other topic is again a complication of IVF, that is multiple pregnancy .It is a case report .

Thanks with best regards
Dr. D’Pankar Banerji

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1. Monitoring in Induction of Ovulation during IUI and IVF

Monitoring can be divide in three stages:
1. Before starting induction therapy
2. During The period of Induction
3. Period that follows completion of therapy


1. Before starting induction therapy : This depends mainly on factors like, patient’s endocrine profile and general health, her age and financial situation and the physician’s previous experience. We discuss various inducing agents in IUI and IVF and their special feature of monitoring

A. Clomiphene citrate : Here FSH should not be abnormally high and patient should not be estrogen deficient ( like feature of ovarian failure ) . Sonography should be done before starting the drug to rule out any ovarian cyst.

B. Gonadotrophin therapy : Ovarian incompetence has to be ruled out first as treatment is costly. Day2 or Day3 FSH helps in that ( <10 or <15 mIU/ml).Non gynecological endocrine problems should be treated first. Hyperprolactiniemia and galctorrhea requires evaluation of intracranial lesions. Hyperprolactinemia has no adverse effect on ovarian response to exogenous gonadotropin therapy. Prior USG to rule out any previous cysts or PCOS.

C. Gonadotropin releasing hormone analogue ( GnRH a) combined with exogenous gonodotropins : This therapy is especially effective for women either show no response to exogenous gonadotropins or who develop premature spontaneous LH and progesterone rise. It prevents premature luteinization which is a major reason for decreased srccesss with other therapies. GnRha are either started in luteal phase of previous cycles( long protocol) or started in follicular phase concomitantly with Gn ( HMG and/or FSH). In long protocol there are criteria of pituitary and ovauan suppression. It is verified by onset of menstration associated wth a serum LH < 2 mIU/ml, estradiol( E2) <50 or <30 pg/ml and by the absence of any ovarian follicles > 10 mm in diameter.

2. During the period of induction : It mainly depends on biophysical parameters of follicular growth and hormonal parameters principally E2 and sonography of endometrium.

A. Biophysical parameters of follicular growth :

(i) Natural cycle : Follicles spontaneously reach maturity ,inner dimensions 17-25 mm. Within the same individual ,the size of a mature follcile is relatively constant. Intrafolliclular echoes may be observed in mature follicles arising from clusters of guanulosa cells that shear off the wall near the time of ovulation.

(ii) CC treated cycles : Each follicle seems to develop at an induvidual rate,and at times may be accelerated or slowed down.Therefore the largest follicle on a given day may not be the same one that is the largest two day later , and may not even be the one that is most mature .Correlation of E2 and follicle size is poor and the maximum pre ovulatory diameter can range from 19-24 mm.

(iii) HMG treated patients : In amenorrhic women with dormant ovaries ( Low pituitary activity or hypothalamic amenorrhea ), a small number of large follicles develop. The growth rate and E2 production are linear, a high pregnancy rate is achieved in this group. In contrast , stimulation of patients with estrogenic activity requires less hMG and usually results in the rapid recruitment of many follicles with different growth rate and E2 secretory capacity. The rate at which E2 increases is exponential, increasing the risk of Hyperstimulation.The growth rates and functional maturity are asynchronous. In this group of women ,both E2 and sonographic follicular monitoring are essential.

Sonographic delineation of follicle size is crucial because hCG is best admistered once follicles reach 15-18 mm in size. For IVF cycles, follicles are typically aspirated when they reach 15-18 mm in average diameter and when the E2 level is approximately 400 pg/ml per large follicle. Another sonographic sign of mature follicle is the presence of low level intrafollicular echoes.

The serum level of E2 on the day of hCG injection is
E2 = 291 pg/ml + 180(x) + 64(y) + 18.7(z)
Where x,y and z represent follicles measuring > 17mm, 15 to 16 mm and < 14 mm respectively ----Ref 1.
Sonographic findings of enlarged ovaries with multiple follicles may suggest the possibility of hyperstimulation ,extremely high level of E2 ( over 3000 pg/ml ) can be more accurate predictor of this syndrome.

B. Sonography of endometrium : Patients who achieve pregnancy more frequently have a multilayered periovulatory endometrium .Endometrium with outer peripheral layer fo dense echogenicity surrounding a central sonolucent area ( hallo pattern ) with more than 9 mm thickness achieve better pregnancy rates.

C. Monitoring E2 levels : poor responder are those when E2 level is < 300 pg/ml on day 8 of stimulation. Dose of gonadotropins should not be changed as long as serial E2 level rise between 50 and 100 % every other day. Very slow or very rapid growth rate have less pregnancy rates. Using GnRha and Gn in a downregulation protocol, the ovarian response was evaluated in terms of E2 levels on the day of hCG injection. Low responders ( <800 pg/ml) medium ( 800-1500 pg/ml) and high responders ( >1500 pg/ml), there was no difference between the three group in respect to development of mature oocytes and rapidly cleaving embryos. The pregnancy rate in the low responding group was significantly lower than in the other two groups .Thus it seems that the receptivity of the endometrium depends at least partially on adequate E2 levels. It also seems that E2 levels do not directly correlate with oocyte maturity and embryonic growth

D. Timing of hCG administration : The dose is between 2000-10,000 IU. Injected on sixth day of sustained increase in serum estradiol levels.In some centers it is injected when E2 level is 200-300 pg/m per follicle < 17-18 mm.

3. Monitoring after the completion of induction therapy :

In IVF oocyte retrieval is done after 35 hours. All the follicles should be aspirated to reduce the chance of hyperstimulation syndrome. Be careful regarding the ovarian torsion .

2. Triplet ,after two blastocyst transfer, (Monozygotic twinning of one blastocyst)—Case report.

Pateint name ,Mrs. S.C. 35 yrs, register at us for IVF .Indication is tubal factor. She was downregulated by luteal phase luprofact daily injection for 15 days . Quiescence of ovaries was identified by sonographic examination of ovaries ( no follicles more than 5mm ) and endometrial thickness ( less than 3 mm ). She was stimulated by FSH hp300 IU for three days and then HMG hp 300 IU for 6 days . Monitoring was done solely by transvaginal sonography. Injection Luprofact was continued till the day of hCG injection. Total 9 oocytes were retrieved . All are inseminated conventionally . 6 fertilized .
On day 3 ,two were 8-10 cells and two were in 6-8 cell stage, rest two arrested at two cell stage.
All are grown to blastocyst stage in a sequential culture in blastocyst media. On day 5 ,there were two very good blastocysts . Both the blastocysts were transferred under USG guide and transfer was smooth and atraumatic on 4th Jan 2008.

Luteal phase support was with vaginal progesterone.
Urine pregnancy test was done on 18th Jan and was positive.
Transvaginal sonography on 29th Jan shows two well defined gestational sac and twin pregnancy was disclosed to the patient. She came back after one month and on 25th Feb 2008 ,sonography was done to see the health of the fetuses and their growth . USG shows two sacs ,but one sac contains two live fetuses and other sac contains another live fetus. It was confirmed that ,it is a triplet pregnancy after two blastocyst transfer ,with monozygotic twinning of one embryo.

Discussion : Blastcyst transfer was aimed to reduce the number of transferred embryos to reduce the chances of multiple pregnancy. It has been found in the literature that prolonged culture of human embryos to blastocyst stage increases the chance of monozygotic twinning. But there are also report that monozygotic twinning are increased even without prolong culure. It was postulated that ,it is the culture condition and not the duration that creates monozygotic twinning.

3. Training in IVF and Embryology
Module I : Ovulation induction and Intra Uterine Insemination ( One day )
Module II : Conventional IVF and fundamentals of Embryology( Two days )
Module III : Intra cytoplasmic sperm injection, Micromanipulation ( Two days)
Course fees :

Module I : Rs.2000.00 ( US$ 50 )
Module II : Rs.20,000.00 ( US$ 500 )
Module III : Rs. 50,000.00 ( US$ 1250 )