This continues our series of case studies on unexplained IVF failure with our sixth case of poor egg/embryo quality in young women with good ovarian reserve. In this case, we profile a couple doing IVF with PGD for Gender Selection.

Background:
When I met Mary she was 34 years old. Both she and her husband had proven fertility. They had over a period of 5 years spontaneously and without difficulty achieved three (3) full term natural deliveries of healthy boys. Thereupon, they desperately wanted to have a little girl and sought the services of their local Reproductive Endocrinologist (RE) to help them achieve their dream. Her RE put her on clomiphene citrate and performedintrauterine insemination (IUI) with husband’s sperm which was first processed in an attempt to bias the odds of their propagating female embryos.

After four (4) back-to back failed attempts to achieve a pregnancy, they were advised to consider IVF. Fertilization would be achieved by injecting each mature egg (MII) by intracytoplasmic sperm injection (ICSI) with female gender biased, processed sperm sample and thereupon to have the resulting embryos subjected to preimplantation genetic diagnosis (PGD) and karyotyped to identify female embryos as well as to screen forchromosomal integrity (“competence”) using fluorescence in situ hybridization (FISH). They would then selectively transfer one or more of the “female” embryos to her uterus.

Mary had perfectly normal ovarian reserve (day-3 blood FSH/LH/ [E2]/AMH= 6.5MIU/ml/2.7 MIU/ml/47pg/ml/ 4.3 respectively) and accordingly underwent three (3) successive IVF attempts using a modest long pituitary protocol of controlled ovarian stimulation (COS). She responded well, and following 9 days of stimulation, she developed 14 follicles, 9 of which measured between 18 and 21mm in diameter. At this point she was “triggered” with 250mg of Ovidrel [a DNA-recombinant form of hCG (hCGr)].

She yielded ten MII eggs at egg retrieval, all of which were successfully fertilized by ICSI. They went on to divide (cleave). However on day 3 post-ICSI, all showed a significant degree of fragmentation (>20%), suggesting that they were of questionable quality. By this time, one (1) embryo had reached the 8 cell stage of cleavage, one (1) was 6 cells and the remaining two (2) were less than 6 cells. All four embryos were all subjected to PGD followed by 12-probe FISH to assess chromosomal integrity (karyotyping).

For FISH to be effective, at least one (1) cell (blastomere) must be removed (biopsied) intact and sent to a reproductive genetics laboratory for karyotyping and gender identification. In this case, biopsies of two (2) of the four (4) embryos failed to yield an intact cell suitable for karyotyping. Accordingly, a 2nd cell had to be biopsied (a rather traumatic development). Karyotyping subsequently revealed that three (3) of the four (4) embryos were female and one (1) was male. Unfortunately, all had numerical chromosomal abnormalities (aneuploidy) and none developed into expanded blastocysts. Thus no ET was performed.

Two (2) months later, a second In Vitro Fertilization attempt was undertaken. The same ovarian stimulation protocol was used, this time yielding five (5) embryos that as in the 1st IVF cycle were again significantly fragmented. Only two (2) of the embryos had cleaved to the 6 -8 cell stage. These two (2) embryos went on to develop in to blastocysts. One was diagnosed as being a chromosomally normal, female embryo and subsequently developed into an expanded blastocyst. This embryo was transferred to Mary’s uterus, but no pregnancy resulted.

The 3rd and final IVF attempt was conducted using the same protocol (with a slightly higher dosage of gonadotropins) and yielded five (5) embryos that were once again fragmented. Three (3) were female embryos and two were male. All three (3) female embryos made it to the blastocyst stage but only one (1) was deemed to be karyotypically normal based upon PGD/FISH. It was transferred to Mary’s uterus. Once again…no pregnancy followed.

Commentary:
Mary was a young patient. She and her husband both had proven fertility. Accordingly, her failure to conceive with four (4) clomiphene-IUI attempts coupled with the fact that 3 IVF attempts consistently produced poor quality embryos requires explanation. The following factors are thus pertinent:

1. IUI using clomiphene: I cannot fault the use of (low cost) clomiphene citrate for IUI, with two the following provisos:

- Patients should be informed in advance of initiating treatment that the success rate using clomiphene is likely to be 20-30% lower than when IUI is performed under gonadotropin stimulation.
- Since clomiphene is an anti-estrogen and builds up in the body over time, it should not be administered for more than 3 consecutive cycles (back-to back) without at least one (1) resting cycle before being used again. If this is ignored it can, by thinning the uterine lining and/or causing dysfunctional ovulation, become somewhat of a contraceptive.

There are both absolute and relative contraindications to the use of clomiphene. These include a) women over 40 years of age and /or those of any age who have diminished ovarian reserve. In such cases there is usually an inability to produce enough follicles to cause the estrogen levels to rise high enough to overcome the anti-estrogen effects of clomiphene; b) women with endometriosiswho uniformly have a “toxic pelvic environment” through which ovulated eggs must pass, thereby profoundly reducing their fertilization potential; c) women with damaged, adhesed orblocked Fallopian tubes (from whatever cause).

2. Sperm processing for gender selection: Based upon significant experience, when it comes to gender selection, available methods for sperm processing fall far short of the mark in my opinion…especially when it comes to gender selection for IVF. There is data to suggest that some methods might well improve gender bias when it comes to IUI…but alas, not when used in IVF. In fact, in my opinion, it is likely that some of the methods of sperm processing for gender selection could so damage the sperm as to increase the yield of “poor quality” embryos and reduce IVF success. Thus, in my view, given these detractors and the current state of the technology, it is preferable to do PGD for gender selection on embryos that have not been propagated from sperm previously processed for gender biasing.

When it comes to the ethics of embryo gender selection, there are several issues to consider:
First is an incontrovertible reality that gender selection has a valuable role to play in preventing certain serious genetic disorders that have a preponderance in either males or females, in order to try and avoid a medical disaster. I submit that in such cases, embryo gender selection is not only a good idea, but is a valuable treatment.

Second is the issue of “convenience gender biasing.” Here, the patient seeks gender selection of embryos solely for the sake of preference. In my practice I will only agree to assist in such cases when the couple has no other children of the chosen gender and simply wants to effect “family balancing.”

3. Laboratory expertise in performing embryo biopsy:Relatively few embryologists perform a sufficient number of embryo biopsies to become proficient at performing this technologically complex procedure. In fact, most only perform a handful of such procedures per year. They should therefore not be doing PGD biopsies without adequate supervision, until they gain the required experience. This appears to have been a possible contributing factor that led to poor embryo quality in Mary’s case. Note that the embryologist had to remove two blastomeres (cells) rather than just one. This was likely to have been very traumatic to the embryo, potentially resulting in compromised embryo development in this case.

Moreover, when sufficient expertise is lacking, the embryo being biopsied has to remain outside the incubator for a long period of time, rendering it highly vulnerable to the elements and compromising its subsequent development.

4. The method used to Karyotype the embryos: FISH cannot reliably access all 23 chromosome pairs. It can only identify 8-12 of the 23 pairs of chromosomes. As such it does not fully karyotype embryos. Accordingly, even when FISH/PGD yields a “normal” diagnosis there is at least a 40% chance that aneuploidy affecting unrecognized chromosomes still exists. Thus today, it is preferable to use comparative genomic hybridization (CGH) which assesses all chromosomes (full karyotyping) and at the same time can also accurately identify the gender of the embryo. This was not done in Mary’s case. I believe it to be highly likely that even those embryos deemed normal by PGD/FISH were actually aneuploid and “incompetent”.

5. The use of Ovidrel (hCGr) to “trigger” egg maturation (meiosis). Ovidrel at a dosage of 250mg is, in my opinion, not sufficiently biologically potent to optimally “trigger meiosis”. In my opinion for Ovidrel to be effective the dosage needs to be increased to 500mg. However, there is no real benefit in using hCGr because 10,000U of urinary-derived hCG (hCGu) such as is available in the form of Profasi, Novarel or Pregnyl, works just as well a 500mg of Ovidrel and costs much less.

Follow Up:
I subsequently treated Mary using an agonist/antagonist conversion protocol (LA-4). She was triggered on day-10 of COS using 10,000U of Profasi. I harvested 11 mature (MII) eggs. Nine (9) were successfully fertilized by ICSI using non-gender processed sperm. PGD with CGH was done on six day-3 embryos that were between six (6) and eight (8) cells cleaved and had virtually no fragmentation. Six developed into expanded, good quality blastocysts and were vitrified (cryobanked) for subsequent dispensation in a later cycle once CGH results were available (“staggered IVF”). Three (3) of these embryos were found to be CGH-normal with two (2) being female.

Subsequently in a later cycle, we thawed (warmed) these twovitrified female embryos and following appropriate hormone replacement, transferred them to Mary’s uterus. She conceived and subsequently gave birth to one healthy little girl.

Addendum: It is important to understand that IVF is an ART-Science blend and not all practitioners agree on the same strategies. Thus, in the final analysis, it is important, after discussion with your personal doctor, to follow his/her advice to the letter.

Feel free to present your case history in the comments and I will do my best to offer my opinion.